Method for producing scyllo-inositol

ABSTRACT

It is intended to provide a novel NAD + -independent myo-inositol 2-dehydrogenase which converts myo-inositol into scyllo-inosose in the absence of NAD + ; a novel enzyme scyllo-inositol dehydrogenase which stereospecifically reduces scyllo-inosose into scyllo-inositol in the presence of NADH or NADPH; and a novel microorganism which belongs to the genus  Acetobacter  or  Burkholderia  and can convert myo-inositol into scyllo-inositol. By using these enzymes or the microorganism, scyllo-inositol is produced. Furthermore, scyllo-inositol is purified by adding boric acid and a metal salt to a liquid mixture containing scyllo-inositol and a neutral saccharide other than scyllo-inositol to fonts a scyllo-inositol/boric acid complex, separating the complex from the liquid mixture, dissolving the thus separated complex in an acid to give an acidic solution or an acidic suspension and then purifying scyllo-inositol from the acidic solution or the acidic suspension.

RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.10/576,030, filed Apr. 13, 2006 which is the U.S. National Phase under35 U.S.C. §371 of International Application PCT/JP2004/015174, filedOct. 14, 2004, which claims priority to JP 2003-353490, filed Oct. 14,2003; JP 2003-353491, filed Oct. 14, 2003; JP 2004-018128, filed Jan.27, 2004; and JP 2004-194088, filed Jun. 30, 2004.

TECHNICAL FIELD

The present invention relates to a method of producing scyllo-inositolfrom myo-inositol by means of microbial conversion.

The present invention also relates to a novel NAD⁺-independentmyo-inositol 2-dehydrogenase and a method of producing the same. Thepresent invention also relates to a method of screening a microorganismfor producing scyllo-inosose based on an activity of NAD⁺-independentmyo-inositol 2-dehydrogenase. The present invention further relates to amethod of producing scyllo-inosose and scyllo-inositol using anNAD⁺-independent myo-inositol 2-dehydrogenase or a strain having a highactivity of said enzyme.

The present invention also relates to a novel enzyme, scyllo-inositoldehydrogenase and a method of producing scyllo-inositol using saidenzyme. Specifically, the present invention relates to a novel enzyme,scyllo-inositol dehydrogenase which catalyzes an oxidation-reductionreaction between scyllo-inositol and scyllo-inosose andstereospecifically reduces scyllo-inosose into scyllo-inositol in thepresence of NADH or NADPH; and a method of producing scyllo-inositolusing the enzyme.

The present invention further relates to a method of efficientlyproducing scyllo-inositol from a liquid mixture containingscyllo-inositol and neutral sugars other than scyllo-inositol.

The scyllo-inositol can be used as a therapeutic agent for treatment ofan Alzheimer disease, a raw material for synthesis of bioactivesubstances, or a raw material for synthesis of liquid crystal compounds.

BACKGROUND ART

Myo-inositol is a naturally-occurring known substance represented by thefollowing steric structural formula (A).

Scyllo-inosose is a known substance represented by the steric structuralformula (B).

Furthermore, scyllo-inositol is a known substance represented by thefollowing steric structural formula (C).

Scyllo-inositol is one of stereoisomers of myo-inositol and is asubstance widely found among animals and plants. Scyllo-inosose is acompound having a structure in which an axial hydroxyl group at thesecond position of myo-inositol is oxidized, and exists generally as anatural compound.

Scyllo-inositol is a substance expected for applications such as atherapeutic agent for an Alzheimer disease (see Non-Patent Document 1),a raw material for synthesis of bioactive substances (Patent Document1), or a raw material for synthesis of liquid crystal compounds (PatentDocument 2).

Examples of a method of producing scyllo-inosose or scyllo-inositol bymeans of a chemical synthetic procedure include: (i) a method ofobtaining scyllo-inositol by reducing hexahydroxybenzene with Raneynickel (Non-Patent Document 2); (ii) a method of obtainingscyllo-inositol by reducing scyllo-inosose obtained from a glucofuranosederivative through a reaction involving five steps (Non-Patent Document3); (iii) a method of obtaining scyllo-inositol using as a raw materialcis-trioxa-tris-homobenzene through a reaction involving four steps ormore (Non-Patent Document 4); and (iv) a method of obtainingscyllo-inositol including oxidizing myo-inositol with a platinumcatalyst to thereby obtain scyllo-inosose, and subjecting thescyllo-inosose to esterification followed by reduction and hydrolysis(see Patent Document 2).

As a method of converting myo-inositol into scyllo-inositol using amicroorganism, a method using a bacterium belonging to the genusAgrobacterium is known (Patent Document 3). However, this method is notapplicable for an industrial-scale production because of low yield ofscyllo-inositol and generation of other converted products.

Meanwhile, a bacterium belonging to the genus Acetobacter (seeNon-Patent Document 5) is known to act on myo-inositol to absorb oxygento thereby oxidize myo-inositol into scyllo-inosose. However, itsdetailed mechanism has not been studied.

The enzyme which oxidizes myo-inositol into scyllo-inosose (myo-inositol2-dehydrogenase) has been reported from a number of organisms such asanimals, algae, yeasts, and bacteria, and it is an enzyme that widelyexists in nature. Examples of a typical microorganism having the enzymeinclude Aerobacter aerogenes (see Non-Patent Document 6), bacteriabelonging to the genus Bacillus (Non-Patent Document 7 and 8; PatentDocuments 4-6), and bacteria belonging to the genus Pseudomonas(Non-Patent Document 9 and 10).

However, the myo-inositol 2-dehydrogenases in those reports areNAD⁺-dependent enzymes, therefore they require NAD⁺ or NADP⁺ foroxidation. When the enzyme is subjected to an industrial-scale reaction,fermentative production must be employed in order to recycle thoseco-enzymes, resulting in decomposition of part of substrates. Inaddition, there had been problems in industrial-scale production suchthat the concentration of the substrate should be kept low.

Meanwhile, there is a report of the presence of a scyllo-inositoldehydrogenase in a bovine brain and a fat tissue of a cockroach(Non-Patent Document 11). When scyllo-inosose as a substrate is reducedby this enzyme with NADPH, both of scyllo-inositol and myo-inositol arereported to be generated. However, the enzyme has low substratespecificity, a highly purified enzyme was not used, and other propertiesare unknown, therefore the enzyme may be an alcohol dehydrogenase havinglow substrate specificity. Therefore, the enzyme has not been describedin Handbook of Enzymes (published by Asakura Shoten). As describedabove, although reports on animals exist, it has not been ascertainedwhether these reports are true.

Furthermore, there is also a known method of producing scyllo-inositolby chemically reducing scyllo-inosose produced by microbial oxidation(Patent Document 7). Since the substance obtained by the chemicalreduction of scyllo-inosose is a mixture of scyllo-inositol andmyo-inositol, the mixture had to be desalted and purified, followed byseparation of scyllo-inositol having low solubility from theconcentrated solution by crystallization. Thus, those methods haverequired many operations and thus there has been a room for improvementwith respect to the yield of scyllo-inositol. Under such circumstances,the development of a method of producing purified scyllo-inositol from amixture of scyllo-inositol and myo-inositol which is obtained byreduction of scyllo-inosose, or the like, has been expected in order toproduce scyllo-inositol conveniently and efficiently.

When scyllo-inosose is reduced using NaBH₄ in a solution, the solutionafter the reaction contains myo-inositol, scyllo-inositol, and a smallamount of a scyllo-inositol/boric acid complex. For suchscyllo-inositol/boric acid complex, there has been known a method ofobtaining scyllo-inositol involving: filtrating the complex as aprecipitate; dissolving the precipitate in diluted sulfuric acid; addingthereto methanol to subject it to azeotropy with boric acid; removingthe boric acid; and desalting the remaining solution using an ionexchange resin (Non-Patent Document 12).

The scyllo-inositol/boric acid complex is a substance represented by thefollowing steric structural formula (D).

However, in the above-described method of reducing scyllo-inosose usingNaBH₄, the ratio of the generated scyllo-inositol/boric acid complex islow, and scyllo-inositol is also generated in the solution. Therefore,the complex and components in the solution had to be separated tothereby obtain scyllo-inositol from each of those. Furthermore, a largeamount of an organic solvent has been required to obtain scyllo-inositolfrom the complex, there has been a room for improvement in an economicalviewpoint. Thus, there have been demanded a method of producingscyllo-inositol conveniently and efficiently in industrial-scaleproduction.

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DISCLOSURE OF THE INVENTION

It is an object of the present invention to provide a method ofproducing scyllo-inositol directly from myo-inositol at high yields bymeans of only microbial conversion.

Furthermore, it is an object of the present invention to provide a novelenzyme for catalyzing a conversion reaction from myo-inositol intoscyllo-inosose, and a novel method of producing scyllo-inosose andscyllo-inositol using the enzyme.

Furthermore, it is an object of the present invention to provide a novelscyllo-inositol dehydrogenase which catalyzes an oxidation-reductionreaction between scyllo-inositol and scyllo-inosose, andstereospecifically reduces scyllo-inosose into scyllo-inositol under thepresence of NADH or NADPH, and a novel method of producingscyllo-inositol using the enzyme.

Furthermore, it is an object of the present invention to provide a novelmethod of efficiently producing scyllo-inositol having high purity froma liquid mixture containing scyllo-inositol and neutral sugars otherthan scyllo-inositol.

The inventors of the present invention have studied to search amicroorganism capable of producing scyllo-inosose from myo-inositol, andhave found a bacterium belonging to the genus Acetobacter which wasseparated from nature (AB10253 strain). The strain was deposited inInternational Patent Organism Depositary, National Institute of AdvancedIndustrial Science and Technology with an Accession number of FERMP-18868 (International depositary number FERM BP-10136). This strain wasused to establish a method of producing scyllo-inosose frommyo-inositol, and a method of producing scyllo-inositol from theobtained scyllo-inosose by means of chemical reduction (JP 2003-102492A).

Subsequently, in order to improve the ability to convert intoscyllo-inosose, the strain was subjected to breeding by mutation. Bythis operation, the inventors have found that there exist some strainswhich biologically reduce scyllo-inosose generated from myo-inositol andgenerate a small amount of scyllo-inositol, among the mutant strains.Then, the inventors of the present invention subjected those strains tobreeding by mutation, in order to obtain a strain which produces andaccumulates mainly scyllo-inositol directly from myo-inositol by meansof only conversion by culture. As a result, they have succeeded inobtaining a mutant strain which meets the object, that is, a strainwhich has acquired an ability to reduce scyllo-inosose generated by thereduction of myo-inositol into scyllo-inositol and accumulating thescyllo-inositol in a medium.

Furthermore, although a conversion ability is lower than that of theAB10253 strain, strains each capable of generating scyllo-inositol frommyo-inositol have been found in nature. Identification using theirnucleotide sequences of 16Sr RNA has confirmed that those strains aremicroorganisms each belonging to Acetobacter cerevisiae, Acetobactermalorum, or Burkholderia andropogonis.

The inventors of the present invention have found that scyllo-inositolcan be efficiently produced by using those microorganisms.

The inventors of the present invention have considered that if an enzymecapable of efficiently converting myo-inositol into scyllo-inosose canbe acquired, efficient production of scyllo-inosose from a highsubstrate concentration of myo-inositol is possible by using suchenzyme. Also, they considered that if a strain having a high activity ofsuch enzyme can be isolated by screening, such strain is able to be usedfor the production of scyllo-inosose.

Under a hypothesis that there is a novel type of NAD⁺-independentmyo-inositol 2-dehydrogenase which has not been known so far and capableof catalyzing a conversion reaction from myo-inositol intoscyllo-inosose, the inventors of the present invention have madeextensive studies to obtain such an enzyme. As a result, they have foundthat NAD⁺-independent myo-inositol 2-dehydrogenase is present inAcetobacter sp. AB10253 strain belonging to the genus Acetobacter. Theinventors of the present invention have succeeded in efficientlyproducing scyllo-inosose by using this enzyme or a microorganism havinga high activity of this enzyme, and in efficiently producingscyllo-inositol with high purity by reducing the obtainedscyllo-inosose.

Next, the inventors of the present invention have studied howmyo-inositol is converted into scyllo-inositol in the microbial cells ofthe strain which produces scyllo-inositol directly from myo-inositol. Asa result, a hydroxyl group at the second position of the myo-inositol isoxidized in an oxygen-dependent manner to generate scyllo-inosose, andthen scyllo-inositol is formed by the function of an enzyme having anactivity of reducing scyllo-inosose into scyllo-inositol in NADH orNADPH-dependent manner. Based on such an activity, the inventors havesucceeded in purifying an enzyme having an activity of reducingscyllo-inosose into scyllo-inositol. They also found that this enzymehas an activity to stereospecifically reduce scyllo-inosose intoscyllo-inositol in an NADPH-dependent manner and an activity to oxidizescyllo-inositol into scyllo-inosose in an NADP⁺-dependent manner, andnamed “scyllo-inositol dehydrogenase”. In addition, this enzyme has anability to oxidize a hydroxyl group at the fifth position ofmyo-inositol, therefore this enzyme can also be referred to asmyo-inositol 5-dehydrogenase.

Furthermore, the inventors have succeeded in cloning of genes eachencoding a scyllo-inositol dehydrogenase by PCR from genomes ofEscherichia coli, the genus Agrobacterium, Bacillus subtilis, andXanthomonas campestris.

Furthermore, since the enzyme performs an oxidation-reduction reactionin an NAD⁺ or NADP⁺-dependent manner, the inventors of the presentinvention considered that scyllo-inositol can be directly converted frommyo-inositol via scyllo-inosose by combining the enzyme and a knownmyo-inositol 2-dehydrogenase (having an activity to reducescyllo-inosose into myo-inositol in NAD⁺ or NADP⁺-dependent manner: EC1.1.1.18) (see: FIG. 1), and they have succeeded.

Moreover, the inventors of the present invention have considered that inorder to produce scyllo-inositol efficiently from a liquid mixturecontaining scyllo-inositol and neutral sugars such as myo-inositol whichis obtained by reduction of scyllo-inosose, it is advantageous to form ascyllo-inositol/boric acid complex. On the basis of such consideration,the inventors of the present invention have extensively studied on amethod of efficiently bringing only scyllo-inositol in a liquid mixturecontaining scyllo-inositol and myo-inositol into such ascyllo-inositol/boric acid complex. As a result, they have found that ascyllo-inositol/boric acid complex consisting of scyllo-inositol, boricacid, and a metal ion has a specific association and is a complex havinglow solubility which is different from other complexes of neutralsugars. Furthermore, the inventors have found that thescyllo-inositol/boric acid complex is effectively formed andprecipitated by: adding boric acid and a metal salt in amounts twicemoles or more, preferably twice to three times more than that ofscyllo-inositol dissolved in a liquid mixture; and maintaining thesolution in an alkaline condition of pH 8.0 to 11.0, preferably pH 9.0to 10.0. Under such conditions, the scyllo-inositol/boric acid complexwas formed from a liquid mixture containing scyllo-inositol and neutralsugars such as myo-inositol. Then, the complex was dissolved in an acid,followed by purification using an ion exchange resin or water-solubleorganic solvent, and thereby scyllo-inositol is efficiently produced.

Thus, the present invention has been completed.

That is, the present invention provides the followings.

(1) A method of producing scyllo-inositol comprising:

allowing a microorganism capable of converting myo-inositol intoscyllo-inositol and belonging to the genus Acetobacter or Burkholderiato react with myo-inositol in a solution containing myo-inositol toproduce and accumulate scyllo-inositol in the solution; and

collecting the scyllo-inositol from the solution.

(2) The method according to (1), wherein the solution containingmyo-inositol is a liquid medium containing myo-inositol, and themicroorganism is allowed to react with myo-inositol by culturing themicroorganism in the liquid medium.

(3) The method according to (1), wherein cells obtained by culturing themicroorganism is allowed to react with myo-inositol in the solution.

(4) The method according to any one of (1) to (3), wherein themicroorganism is a microorganism belonging to Acetobacter cerevisiae,Acetobacter malorum, or Burkholderia andropogonis.

(5) The method according to any one of (1) to (3), wherein themicroorganism is Acetobacter sp. AB10281 strain (FERM BP-10119) or amutant strain thereof.

(6) Acetobacter sp. AB10281 strain (FERM BP-10119) or a mutant strainthereof having an ability to convert myo-inositol into scyllo-inositol.

(7) NAD⁺-independent myo-inositol 2-dehydrogenase having at least thefollowing physiological properties:

(a) Action: catalyzing a reaction that deprives myo-inositol of electronto produce scyllo-inosose in the presence of an electron acceptingsubstance

(b) Optimum pH: the activity is maximum at pH of 4.5 to 5.5

(c) Cofactor: containing 1 mol of heme iron per 1 mol of the enzyme

(d) Inhibitor: the activity of the enzyme is inhibited to 1% or lower by1 mM of Sn²⁺ ion

(e) Subunit structure: a heteromer at least comprising proteins eachhaving a molecular weight of 76 k Dalton or 46 k Dalton

(g) Substrate specificity: acting on D-chiro-inositol, muco-inositol,and myo-inositol to convert them into D-chiro-1-inosose,L-chiro-2-inosose, and scyllo-inosose, respectively, but not acting onallo-inositol, scyllo-inositol, L-chiro-inositol, and glucose

(8) A method for producing myo-inositol 2-dehydrogenase, comprising:

culturing a microorganism which has an ability to produceNAD⁺-independent myo-inositol 2-dehydrogenase and belongs to the genusAcetobacter; and

separating and purifying the myo-inositol 2-dehydrogenase from the cellsof the cultured microorganism.

(9) The method according to (8), wherein the microorganism isAcetobacter sp. AB10253 strain (FERM BP-10136).

(10) A method for producing scyllo-inosose, comprising:

generating scyllo-inosose by allowing NAD⁺-independent myo-inositol2-dehydrogenase to react with myo-inositol in a solution containingmyo-inositol and an electron acceptor; and

separating and purifying the generated scyllo-inosose from the solution.

(11) A method for producing scyllo-inositol, comprising:

generating scyllo-inosose by allowing NAD⁺-independent myo-inositol2-dehydrogenase to react with myo-inositol in a solution containingmyo-inositol and an electron acceptor;

generating scyllo-inositol by allowing the scyllo-inosose to react witha reducing agent; and

separating and purifying the scyllo-inositol.

(12) A method for screening a microorganism for producingscyllo-inosose, comprising:

subjecting Acetobacter sp. AB10253 strain (FERM BP-10136) to amutagenesis treatment to obtain mutant strains; and

selecting a strain from the mutant strains based on NAD⁺-independentmyo-inositol 2-dehydrogenase activity.

(13) A method for screening a microorganism for producingscyllo-inosose, comprising:

isolating microorganisms from a natural sample containing themicroorganisms;

selecting a microorganism from the isolated microorganisms based onNAD⁺-independent myo-inositol 2-dehydrogenase activity.

(14) A method for producing scyllo-inosose, comprising:

generating scyllo-inosose from myo-inositol by culturing themicroorganism for producing scyllo-inosose obtained by the screeningmethod according to (12) or (13) in a medium containing myo-inositol;and

separating and isolating the generated scyllo-inosose from the medium.

(15) A method for producing scyllo-inositol, comprising:

generating scyllo-inosose from myo-inositol by culturing themicroorganism for producing scyllo-inosose obtained by the screeningmethod according to (12) or (13) in a medium containing myo-inositol;

generating scyllo-inositol by allowing the scyllo-inosose to react witha reducing agent; and

separating and isolating the generated scyllo-inositol from the medium.

(16) A scyllo-inositol dehydrogenase having the following physiologicalproperties:

Reaction: as shown in the following formula, catalyzing anoxidation-reduction reaction between scyllo-inositol and scyllo-inososeand stereospecifically reducing scyllo-inosose to scyllo-inositol in thepresence of NADH or NADPH

(17) The scyllo-inositol dehydrogenase according to (16), further havingthe following physiological properties:

(1) Molecular weight and association property: 38 to 46 k Dalton,forming a dimer or a trimer

(2) Coenzyme: requiring NAD⁺ or NADP⁺, or NADH or NADPH as a coenzyme

(3) Activating heavy metals: activated in the presence of Co²⁺ ion

(4) Inhibiting heavy metals: inhibited in the presence of Sn²⁺ ion

(5) Optimum pH: having an activity at pH of 5 to 9

(18) A protein represented by the following (A) or (B):

(A) A protein comprising an amino acid sequence of SEQ ID NO: 28, or

(B) A protein comprising an amino acid sequence of SEQ ID NO: 28,whereby one or plural of amino acids are substituted, deleted, inserted,and/or added, and catalyzing the oxidation-reduction reaction betweenscyllo-inositol and scyllo-inosose and stereospecifically reducingscyllo-inosose into scyllo-inositol in the presence of NADH or NADPH.

(19) A DNA encoding a protein represented by the following (A) or (B):

(A) A protein comprising an amino acid sequence of SEQ ID NO: 28, or

(B) A protein comprising an amino acid sequence of SEQ ID NO: 28,whereby one or plural of amino acids are substituted, deleted, inserted,and/or added, and catalyzing the oxidation-reduction reaction betweenscyllo-inositol and scyllo-inosose and stereospecifically reducingscyllo-inosose into scyllo-inositol in the presence of NADH or NADPH.

(20) A DNA represented by the following (a) or (b):

(a) A DNA comprising a coding region of the nucleotide sequence of SEQID NO: 27, or

(b) A DNA which hybridizes under stringent conditions with a DNA havingthe nucleotide sequence of SEQ ID NO: 27 or a nucleotide sequencecomplementary thereto, and encodes a protein that catalyzes theoxidation-reduction reaction between scyllo-inositol and scyllo-inososeand stereospecifically reduces scyllo-inosose into scyllo-inositol.

(21) A vector comprising the DNA according to (19) or (20).

(22) A transformant microorganism comprising the DNA according to (19)or (20) or the vector according to (21).

(23) The transformant microorganism according to (22), wherein a host tobe transformed is Escherichia coli.

(24) A method for producing scyllo-inositol dehydrogenase, comprising:

culturing the transformant microorganism according to (22) or (23); and

collecting scyllo-inositol dehydrogenase from the culture productthereof.

(25) A method for producing scyllo-inositol dehydrogenase, comprising:subjecting myo-inositol as a substrate to an oxidation conversionreaction into scyllo-inositol at pH 6.0 to 8.5 in the presence of NAD⁺or NADP⁺, in a solution which contains the scyllo-inositol dehydrogenaseaccording to (16) and myo-inositol dehydrogenase (EC 1.1.1.18) whichcatalyzes a reaction of oxidizing myo-inositol to generatescyllo-inosose in the presence of NAD⁺ or NADP⁺.

(26) The method according to (25), wherein scyllo-inositol is added at0.01 to 3% into the solution.

(27) The method according to (25), wherein scyllo-inositol is added at0.2 to 0.5% into the solution.

(28) The method according to (25), wherein cobalt salt and/or magnesiumsalt is added at 0.01 to 5.0 mM into the solution.

(29) The method according to (25), wherein cobalt salt and/or magnesiumsalt is added at 0.2 to 2.0 mM into the solution.

(30) The method according to (25), wherein the concentration ofmyo-inositol in the solution is adjusted to 5 to 22%; and wherein thescyllo-inositol which is generated by the enzymatic reaction iscrystalyzed in the reaction solution, and is separated as a crystal fromthe reaction system by filtration.

(31) The method according to (25), wherein the scyllo-inositoldehydrogenase is a protein represented by the following (A) or (B):

(A) A protein comprising an amino acid sequence of SEQ ID NO: 28, or

(B) A protein comprising an amino acid sequence of SEQ ID NO: 28,whereby one or plural of amino acids are substituted, deleted, inserted,and/or an added, and catalyzing the oxidation-reduction reaction betweenscyllo-inositol and scyllo-inosose and stereospecifically reducingscyllo-inosose into scyllo-inositol in the presence of NADH or NADPH.

(32) The method according to (25), wherein the scyllo-inositoldehydrogenase is a protein encoded by the DNA represented by thefollowing (a) or (b):

(a) A DNA comprising a coding region of the nucleotide sequence of SEQID NO: 27, or

(b) A DNA which hybridizes under stringent conditions with a DNA havinga nucleotide sequence of SEQ ID NO: 27 or a nucleotide sequencecomplementary thereto, and encodes a protein which catalyzes theoxidation-reduction reaction between scyllo-inositol and scyllo-inososeand stereospecifically reduces scyllo-inosose into scyllo-inositol.

(33) The method according to (25), wherein the scyllo-inositoldehydrogenase is a protein represented by the following (C) or (D):

(C) A protein comprising an amino acid sequence of SEQ ID NO: 2, 4, 6,8, 10, 12, or 14, or

(D) A protein comprising an amino acid sequence of SEQ ID NO: 2, 4, 6,8, 10, 12, or 14, whereby one or plural of amino acids are substituted,deleted, inserted, and/or added, and catalyzing the oxidation-reductionreaction between scyllo-inositol and scyllo-inosose andstereospecifically reducing scyllo-inosose into scyllo-inositol in thepresence of NADH or NADPH.

(34) The method according to (25), wherein the scyllo-inositoldehydrogenase is a protein encoded by the DNA represented by thefollowing (c) or (d):

(c) A DNA comprising a coding region of the nucleotide sequence of SEQID NO: 1, 3, 5, 7, 9, 11, or 13, or

(d) A DNA which hybridizes under stringent conditions with a DNA havingthe nucleotide sequence of SEQ ID NO: 1, 3, 5, 7, 9, 11, or 13 or anucleotide sequence complementary thereto, and encodes a protein whichcatalyzes the oxidation-reduction reaction between scyllo-inositol andscyllo-inosose and stereospecifically reduces scyllo-inosose intoscyllo-inositol.

(35) A method for producing a purified scyllo-inositol, comprising:

a first step of forming a scyllo-inositol/boric acid complex by addingboric acid and a metal salt into a liquid mixture containingscyllo-inositol and neutral sugar other than scyllo-inositol in anamount two times or more larger than that of scyllo-inositol dissolvedin the liquid mixture, and by adjusting the pH of the liquid mixture to8.0 to 11.0;

a second step of separating the complex from the liquid mixture;

a third step of dissolving the separated complex into acid to cleaveinto scyllo-inositol and boric acid; and

a fourth step of isolating and purifying the scyllo-inositol from theacidic solution or acidic suspension obtained from the third step.

(36) The method according to (35), wherein, in the first step, theamounts of the boric acid and metal salt to be added is not less thantwice mol, and not more than three times of the scyllo-inositoldissolved in the liquid mixture.

(37) The method according to (35), wherein, in the first step, pH of theliquid mixture is adjusted to 9.0 to 10.0.

(38) The method according to (35), wherein the metal salt to be added isone or more kinds of metal salts selected from the group consisting ofNaCl, NaHCO₃, Na₂CO₃, Na₂SO₄, NaHSO₄, NaH₂PO₄, Na₂HPO₄, Na₃PO₄, borax,KCl, KHCO₃, K₂CO₃, K₂SO₄, KHSO₄, KH₂PO₄, K₂HPO₄, K₃PO₄, MgCl₂, MgCO₃,and MgSO₄.

(39) The method according to (35), wherein the liquid mixture containingthe scyllo-inositol and the neutral sugar other than scyllo-inositol isa liquid mixture containing myo-inositol and scyllo-inositol obtained byreducing scyllo-inosose in a solution containing scyllo-inosose.

(40) The method according to (35), wherein, in the third step, thesolution obtained by dissolving the complex in acid is adjusted to anacidic solution of 0.1 N or higher; and, in the fourth step, the acidicsolution is contacted with an strong acidic ion exchange resin, and witha strong basic ion exchange resin or a boric acid-selective adsorbingresin, and then scyllo-inositol is precipitated from the acidicsolution.

(41) The method according to (35), wherein, in the fourth step,scyllo-inositol is precipitated by adding an aqueous organic solvent tothe acidic solution or acidic suspension.

(42) The method according to (41), wherein the aqueous organic solventis ethanol or methanol; and the ethanol is added in a volume 0.3 to 3times larger, or the methanol is added in a volume 0.3 to 5 timeslarger, than that of the acidic solution or acidic suspension.

(43) The method according to (41), wherein the aqueous organic solventis ethanol or methanol; and the ethanol is added in a volume 0.6 to 1.5times larger, or the methanol is added in a volume 0.9 to 2 timeslarger, than that of the acidic solution or the acidic suspension.

(44) A method of producing scyllo-inositol, comprising:

a first step of obtaining a liquid mixture containing myo-inositol andscyllo-inositol by reducing scyllo-inosose using a metal salt of boronhydride in a solution containing scyllo-inosose;

a second step of dissolving a scyllo-inositol/boric acid complex in theliquid mixture by adding an acid to the liquid mixture and adjusting thesolution to be an acidic solution of 0.01 N or more; and

a third step of precipitating only scyllo-inositol by adding an aqueousorganic solvent to the acidic solution in an amount such that themyo-inositol is not precipitated.

(45) The method according to (44), wherein, in the third step, theaqueous organic solvent to be added is ethanol, methanol, or 1-propanol;and the ethanol is added in a volume 0.2 to 0.4 times larger, themethanol is added in a volume 0.2 to 0.8 times larger, or the 1-propanolis added in a volume 0.2 to 0.4 times larger, than that of the acidicsolution.

(46) The method according to (44), wherein, in the third step, theaqueous organic solvent to be added is ethanol, methanol, or 1-propanol;and the ethanol is added in a volume 0.35 to 0.45 times larger, themethanol is added in a volume 0.45 to 0.55 times larger, or the1-propanol is added in a volume 0.35 to 0.45 times larger, than that ofthe acidic solution.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a schematic view of the principle of the production ofscyllo-inositol by combination of enzymes.

DESCRIPTION OF THE PREFERRED EMBODIMENTS 1. Method of ProducingScyllo-inositol Using a Microorganism Belonging to the Genus Acetobacteror the genus Burkholderia

One embodiment of the present invention relates to a method of producingscyllo-inositol comprising producing and accumulating scyllo-inositol ina solution containing myo-inositol by reacting the myo-inositol with amicroorganism which belongs to the genus Acetobacter or the genusBurkholderia and has an ability to convert myo-inositol intoscyllo-inositol; and collecting the scyllo-inositol from the solution.

The microorganism to be used in the producing method of the presentinvention is a microorganism which belongs to the genus Acetobacter orthe genus Burkholderia and has an ability to convert myo-inositol intoscyllo-inositol. Here, examples of the microorganism belonging to thegenus Acetobacter include Acetobacter cerevisiae, Acetobacter malorum,Acetobacter orleanensis, Acetobacter indonesiensis, Acetobacterorientalis, Acetobacter aceti, Acetobacter liquefaciens, Acetobacterpasteurianus, Acetobacter hansenii, and unidentified strains (sp.)thereof. Examples of the microorganism belonging to the genusBurkholderia include Burkholderia andropogonis, Burkholderiacaryophylli, and Burkholderia graminis. Of those, Acetobactercerevisiae, Acetobacter malorum, and Burkholderia andropogonis areparticularly preferable. The phrase “an ability to convert myo-inositolinto scyllo-inositol” refers to, for example, an ability of amicroorganism to accumulate scyllo-inositol in a medium when themicroorganism is cultured in a medium containing myo-inositol.

A specific example of the microorganism includes Acetobacter sp. AB10281strain. The strain is a strain obtained by mutating Acetobacter sp.AB10253 strain (FERM BP-10136) to impart an ability to convertmyo-inositol into scyllo-inositol. The strain has been deposited inInternational Patent Organism Depositary, the National Institute ofAdvanced Industrial Science and Technology (postal code: 305-8566,Tsukuba Central 6, 1-1, Higashi 1-chrome, Tsukuba, Ibaraki, Japan) withan accession number of FERM P-19639 on Jan. 20, 2004, and is thenconverted to the international deposition under the Budapest Treaty andgiven an accession number of FERM BP-10119.

The Acetobacter sp. AB10253 strain has been deposited in InternationalPatent Organism Depositary, the National Institute of AdvancedIndustrial Science and Technology with an accession number of FERMP-18868 on May 24, 2002, and then converted to the internationaldeposition under the Budapest Treaty and deposited with an accessionnumber of FERM BP-10136.

Derivative strains of a microorganism belonging to the genus Acetobactersuch as Acetobacter sp. AB10281 strain or Acetobacter sp. AB10253 straincan also be used. Such derivative strains can be obtained by mutatingthe microorganism and selecting a strain having a property toselectively and directly convert myo-inositol into scyllo-inositol amongthe strains into which mutation had been introduced. Examples of amutation method include a physical mutation method such as UVirradiation or radiation irradiation, as well as a chemical mutationmethod which employs mutating agents including N-nitrosoguanidine, ethylmethane sulfonate, nitrite, methyl methane sulfonate, an acridine dye,benzopyrene, dimethyl sulfate, and the like.

An example of a method of reacting the above-described microorganismwith myo-inositol in a solution containing the myo-inositol includes amethod of culturing the microorganism of the present invention in aliquid medium containing myo-inositol.

In this case, the composition of the liquid medium to be used is notparticularly limited as long as the object of the present invention isachieved. A liquid medium may be any medium which contains myo-inositolas a material to be converted into scyllo-inositol, in addition tocarbon sources, nitrogen sources, organic nutrients, inorganic salts,and the like. Both a synthetic medium and a natural medium can be used.The liquid medium is added with 0.1% to 40%, more preferably 10% to 30%of myo-inositol, and is preferably added with: 0.1% to 20%, morepreferably 0.3% to 5% of glycerol, sucrose, maltose, or a starch as acarbon source; and 0.01% to 5.0%, preferably 0.5% to 2.0% of a yeastextract, peptone, casamino acid, ammonium sulfate, ammonium chloride,ammonium nitrate, urea, or the like as a nitrogen source. In addition,if necessary, inorganic salts each capable of forming an ion of sodium,potassium, calcium, magnesium, cobalt, manganese, zinc, iron, copper,molybdenum, phosphoric acid, sulfuric acid, or the like can be added tothe medium. A hydrogen-ion concentration in the culture solution doesnot particularly need to be controlled. However, scyllo-inositol isefficiently produced by culturing at conditions adjusted to preferablypH of 4 to 10, more preferably 5 to 9.

Culture conditions vary depending on a kind of the medium. However, aculture temperature is 12 to 35° C., preferably 20 to 27° C. A culturemay be performed aerobically by, for example, shaking the liquid mediumor aerating air or an oxygen gas into the liquid medium. During a mainculture, myo-inositol is oxidized at an early stage of the culture togenerate scyllo-inosose and then the scyllo-inosose is reduced in aliving body at a later stage, to thereby generate scyllo-inositol. Afurther culture results in gradual decomposition of the scyllo-inositol.Therefore, a culture period may be until when the accumulation of thescyllo-inositol becomes the maximum or required amount, and is generally1 to 10 days, preferably 3 to 8 days.

Alternatively, cells of a microorganism obtained by culture may bereacted with myo-inositol in a solution containing myo-inositol. Here,for cells obtained by culture, cells obtained from a microorganismcultured under other appropriate culture conditions may be used, orcells separated and collected from the culture broth of a microorganismused for the production of scyllo-inositol may be reused. The collectionof the cells to obtain the cells may be performed by a known method suchas centrifugal separation or filtration.

By reacting the microorganism with myo-inositol as described above,scyllo-inositol accumulates in the solution. A general method ofisolating and purifying a normal aqueous neutral substance can beapplied for the method of collecting scyllo-inositol from the culturesolution. That is, supernatants of the culture solution are treated withactivated carbon, an ion exchange resin, or the like after cells hadbeen removed from the culture solution, to thereby allow most impuritiesother than scyllo-inositol to be removed. After that, a substance ofinterest can be isolated by using such a method as recrystallization.

More specifically, a culture supernatant in which scyllo-inositol isaccumulated is passed through a column filled with a strong acid cationexchange resin such as Duolite® C-20 (H⁺ type) to remove undesirablecomponents. Flow-through solution is collected, and then deionized wateris passed through the column to wash the column, to thereby collect awash solution. The flow-through solution and the wash solution arecombined together. Then, thus obtained solution is passed through acolumn filled with a strong base anion exchange resin such as Duolite®A116 (OH⁻ type). Flow-through solution is collected, and then deionizedwater is passed through the column to wash the column, to therebycollect a wash solution. The flow-through solution and wash solution arecombined together, to obtain an aqueous solution containingscyllo-inositol but almost no other impurities. The aqueous solution wasconcentrated to thereby obtain a concentrated solution ofscyllo-inositol. The concentrated solution was then added with anappropriate amount of ethanol and left overnight at room temperature ora low temperature, to thereby allow a pure scyllo-inositol crystal to becrystallized. Meanwhile, based on a low water-solubility ofscyllo-inositol, a pure scyllo-inositol crystal can be crystallized justby concentrating and filtrating the aqueous solution. Further, a columnfilled with activated carbon can be used for decolorization, during thecolumn operation.

A pure scyllo-inositol crystal can be obtained by other purificationmethod, such as described later, comprising: preparing ascyllo-inositol/boric acid complex by adding boric acid and NaCl to thesolution containing scyllo-inositol obtained by culture; filtrating andseparating the scyllo-inositol/boric acid complex; allowing boric acidto be released by adding an acid; and crystallizing scyllo-inositol byadding an organic solvent such as methanol.

Furthermore, during the culture of the strain of the present invention,scyllo-inositol having a low water-solubility (solubility of about 1.6%)at normal temperature crystallizes and precipitates. Therefore,myo-inositol can be additionally added during the culture to furtheraccumulate the scyllo-inositol as crystals.

2. Novel NAD⁺-Independent Myo-inositol 2-dehydrogenase, and a Method ofProducing Scyllo-inosose and Scyllo-inositol Using the Enzyme

Other embodiment of the present invention relates to a novelNAD⁺-independent myo-inositol 2-dehydrogenase, and a method of producingscyllo-inosose and scyllo-inositol using the enzyme.

<2-1> Novel NAD⁺-Independent Myo-inositol 2-dehydrogenase

NAD⁺-independent myo-inositol 2-dehydrogenase of the present inventionhas at least the following physiological properties.

(a) Action: catalyzing a reaction that deprives an electron frommyo-inositol to generate scyllo-inosose in the presence of an electronaccepting substance;

(b) Optimum pH: its activity is maximum at pH of 4.5 to 5.5;

(c) Cofactor: requiring 1 mol of hemoferrum per 1 mol of the enzyme;

(d) Inhibitor: its enzymatic activity is inhibited to 1% or lower by 1mM of Sn²⁺ ion;

(e) Subunit structure: a heteromer at least comprising proteins eachhaving a molecular weight of 76 k Dalton and 46 k Dalton;

(g) Substrate specificity: reactive to D-chiro-inositol, muco-inositol,and myo-inositol, and converts them into D-chiro-1-inosose,L-chiro-2-inosose, and scyllo-inosose, respectively, and not reactive toallo-inositol, scyllo-inositol, L-chiro-inositol, and glucose.

The action (a) can be confirmed by determining the myo-inositol2-dehydrogenase activity in the presence of an electron acceptingsubstance. Examples of the electron accepting substance as used hereininclude oxidized DCIP, phenazine methosulfate (PMS), methylene blue, andFe³⁺ ion. Those can be used in combination, however, oxidized DCIP ispreferably used. The myo-inositol 2-dehydrogenase activity can bemeasured defining as 1 unit the activity at which 1 μmol of myo-inositolis oxidized per 1 minute when a reaction rate is calculated based on achange in absorption at 600 nm in a 1 mL solution containing 100 mMphosphate buffer (pH 5.0), 5 mg of myo-inositol, and 0.4 mg of2,4-dichloroindophenol (oxidized DCIP). The optimum pH (b) can beconfirmed by: measuring the activity of myo-inositol 2-dehydrogenase atdifferent pH; and determining the range of pH where the enzymaticactivity shows the maximum value. Meanwhile, the property shown in (d)can be confirmed by comparing the enzymatic activity under which Sn²⁺ion is added to an enzymatic activity determining system, with theactivity under which no Sn²⁺ ion is added thereto. Further, the subunitstructure (e) can be confirmed by SDS-PAGE (sodium dodecylsulfate-polyacrylamide gel electrophoresis) or the like. The molecularweights of 76 k Dalton and 46 k Dalton of respective subunits areapproximate values, and may be around 76 k Dalton and 46 k Dalton.

An example of the NAD⁺-independent myo-inositol 2-dehydrogenase of thepresent invention includes one derived from Acetobacter sp. AB10253strain, but not limited to this one as long as it has theabove-described properties. The substrate specificity of theNAD⁺-independent myo-inositol 2-dehydrogenase of the present inventionis as described above. The specific activity and Km value of the enzymederived from Acetobacter sp. AB10253 strain at a substrate concentrationof 50 mM are represented as follows. That is, D-chiro-inositol (specificactivity of 100%, Km=8.8 mM), muco-inositol (specific activity of 68%,Km=14.5 mM), and myo-inositol (specific activity of 53%, Km=20 mM).

The NAD⁺-independent myo-inositol 2-dehydrogenase of the presentinvention is a NAD⁺-independent myo-inositol 2-dehydrogenase which is atype different from a conventionally known NAD⁺-dependent myo-inositol2-dehydrogenase. The following Table 1 shows the comparison ofparticular differences between both enzymes.

TABLE 1 Enzyme of the present invention Conventional enzyme(NAD⁺-independent type) (NAD⁺-dependent type) Intracellular Membranefraction Cytoplasmic soluble localization fraction Optimum pH pH 4.5-5.5pH 8.0-9.0 Electron Hemoferrum NAD⁺ acceptor<2-2> Method of Producing NAD⁺-Independent Myo-inositol 2-dehydrogenase

A method of producing the NAD⁺-independent myo-inositol 2-dehydrogenaseof the present invention is a producing method comprising culturing amicroorganism which is capable of producing the NAD⁺-independentmyo-inositol 2-dehydrogenase and belongs to the genus Acetobacter, andseparating and purifying myo-inositol 2-dehydrogenase from the culturedcells of the microorganism.

An example of the microorganism which can be used for production of theNAD⁺-independent myo-inositol 2-dehydrogenase includes Acetobacter sp.AB10253 (FERM BP-10136), but not limited to this one as long as it iscapable of producing the NAD⁺-independent myo-inositol 2-dehydrogenase.A conventionally known medium which is used for general culture of amicroorganism and contains a carbon source, a nitrogen source, othernutrients, and the like can be used as a medium for culturing themicroorganism. Here, examples of the carbon source include glucose,sucrose, maltose, and a starch. Preferably, the concentration of thecarbon source to be added is 0.1% to 20%, more preferably 0.3% to 5%.Examples of the nitrogen source include peptone, yeast extract, casaminoacid, ammonium sulfate, ammonium chloride, ammonium nitrate, urea, andmeat extract. Preferably, the concentration of the nitrogen source to beadded is 0.01% to 5.0%, preferably 0.5% to 2.0%. In addition, ifnecessary, inorganic salts each capable of generating an ion of sodium,potassium, calcium, magnesium, cobalt, manganese, zinc, iron, copper,molybdenum, phosphoric acid, sulfuric acid, or the like are preferablyadded to the medium. The expression of the enzyme of the presentinvention is efficiently induced by culturing it in a culture solutionadjusted to pH of 3 to 10, preferably pH of 5 to 7. The value shown by“%” indicates a percentage of w/v, and a concentration shown by “%” alsoindicates the same meaning hereinafter.

In order to induce the expression of NAD⁺-independent myo-inositol2-dehydrogenase, a medium containing myo-inositol is preferably used. Inthis case, myo-inositol is appropriately added at a concentration of0.2% to 15%, preferably 1% to 5%, more preferably 3%.

Culture conditions vary depending on a kind of the medium. However, aculture temperature is preferably 12 to 35° C., more preferably 20 to27° C. Culture is preferably performed aerobically by, for example,shaking the liquid medium or aerating air or an oxygen gas into theliquid medium. A culture period may be preferably up to the day on whichthe myo-inositol completely is eliminated from the culture solution andthe accumulation of the scyllo-inositol becomes the maximum, and isgenerally 1 to 10 days, preferably 3 to 8 days.

The enzyme of the present invention can be obtained by separating andpurifying the enzyme from the cultured cells. The separation andpurification of the enzyme can be performed similarly as a conventionalpurification method of a protein. Specific examples of the separationand purification method is described hereinafter, however, the method isnot limited to these.

First, cells obtained from the culture are precipitated or concentratedby means of centrifugal separation, filtration, or the like. Next, theobtained precipitate or suspension of the cells is disrupted. Frenchpress, dynamill, ultrasonication, or the like can be used for thedisruption, however, ultrasonication is preferable. For example, cellscollected from 1 L of a culture solution are washed with water and arefinally suspended into 50 ml of water. The suspension is subjected toultrasonic to disrupt the cells and then subjected to centrifugalseparation at 12,000 rpm to thereby obtain a precipitate. Subsequently,the obtained precipitate is suspended in an appropriate buffer such asTris buffer or phosphate solution (concentration of 2 mM to 100 mM, pHof 6.0 to 8.0). Then, a surfactant is added thereto, to thereby allow amembrane enzyme to be extracted. Examples of the surfactant includeTriton X-100®, Tween 20®, and Tween 80®. Each of the surfactants can beused at a concentration of 0.02% to 1.0%, however, it is preferable touse Triton X-100 at a concentration of 0.6%.

The enzyme can be extracted by incubating the suspension obtained aboveinto which a surfactant is added, at a low temperature for about 1 to 5hours. Then, the suspension is again subjected to centrifugalseparation, to thereby obtain an enzyme solubilized in a supernatant.Thus obtained enzyme solution can be used for the production ofscyllo-inositol as it is, or if necessary, the enzyme can beconcentrated by a method to be used for general enzyme concentration.Examples of the method of concentrating an enzyme include ammoniumsulfate fractionation and ultrafiltration. Also, the following treatmentis preferably performed in order to purify the enzyme with higherpurity.

The solubilized enzyme is preferably subjected to column chromatographypurification. An example of the column chromatography includes DEAEcolumn chromatography. Any DEAE column containing a DEAE group can beused even if it has different carrier characteristics. A preferableexample of the DEAE column includes a DEAE Toyopearl (manufactured byTosoh Corporation). In the case of purifying the enzyme using a DEAEToyopearl, the enzyme solution may be adjusted to have a saltconcentration of 20 mM before being added to the column. Next, theprotein that had been adsorbed to the column in such a manner is elutedby passing a solution of 20 mM buffer (pH of 6.0 to 8.0) with nosurfactant and with linear concentration gradient of NaCl or KCl. Aconcentration gradient of 0 mM to 500 mM is used for NaCl and aconcentration gradient of 0 mM to 350 mM is used for KCl. Next, 20 mMbuffer (pH of 6.0 to 8.0) with no surfactant is again passed through thecolumn to wash the column, then solution of 20 mM buffer (pH of 6.0 to8.0) with a surfactant and with linear concentration gradient of NaCl orKCl is passed through the column, to thereby elute the protein. Aconcentration gradient of 0 mM to 500 mM is used for NaCl and aconcentration gradient of 0 mM to 350 mM is used for KCl. Examples ofthe surfactant to be added include Triton X-100, Tween 20, and Tween 80.Each of the surfactants can be used at a concentration of 0.02% to 1.0%,however it is preferable to use Triton X-100 at a concentration of 0.1%.Under such conditions, the enzyme of the present invention is elutedfrom the column by means of the 20 mM buffer containing a surfactant and100 to 170 mM of NaCl. Thus obtained enzyme solution can be used for theproduction of scyllo-inositol as it is, or the enzyme solution isfurther treated for higher purification.

In the case of purification based on the enzymatic activity, theenzymatic activity can be measured, for example, by calculating areaction rate based on the change in absorption at 600 nm in 1 mLsolution containing 100 mM phosphate buffer (pH 5.0), 5 mg ofmyo-inositol, and 0.4 mg of 2,4-dichloroindophenol (oxidized DCIP), anddefining the activity to oxidize 1 μmol of myo-inositol per 1 minute as1 unit.

In the case of further purification of the enzyme of the presentinvention, the enzyme solution of the present invention is preferablyadded to, for example, a hydroxyapatite column after being desalted bydialysis or ultrafiltration. In this case, the protein that had beenadsorbed to the hydroxyapatite column is eluted by passing through aphosphate buffer (pH 7.0) with a liner concentration gradient. Theconcentration gradient of the phosphate buffer to be used is 0 mM to 500mM. Examples of the surfactant include Triton X-100, Tween 20, and Tween80. Each of the surfactants can be used at a concentration of 0.02% to1.0%, however, it is preferable to use Triton X-100 at a concentrationof 0.1%. Under such conditions, the enzyme of the present invention iseluted from the column by means of 210 to 260 mM phosphate buffercontaining a surfactant. Thus obtained enzyme solution contains almostpure NAD⁺-independent myo-inositol 2-dehydrogenase, and therefore, itcan be used for the production of scyllo-inositol as it is.

<2-3> Method of Screening a Microorganism for Producing Scyllo-inosose

The present invention also relates to a method of screening amicroorganism for producing scyllo-inosose, comprising mutating andtreating the Acetobacter sp. AB10253 strain; and selecting amicroorganism among the obtained mutant strains based onNAD⁺-independent myo-inositol 2-dehydrogenase activity.

The NAD⁺-independent myo-inositol 2-dehydrogenase activity can bedetermined by, for example, adding a membrane fraction which is obtainedfrom the cells of the microorganism to 1 mL solution containing 100 mMphosphate buffer (pH 5.0), 5 mg of myo-inositol, and 0.4 mg of2,4-dichloroindophenol (oxidized DCIP), and determining the change inabsorption at 600 nm of the resultant. For example, as a criteria ofselection, a strain that exhibits NAD⁺-independent myo-inositol2-dehydrogenase activity 1.2 times or more, preferably twice or morethan that of a non-mutant AB10253 strain is preferably selected, whenthe activity is determined by means of the above-described method andcompared.

For the method of mutating the Acetobacter sp. AB10253 strain, a generalmutating method for a microorganism can be used. Examples of the methodinclude a physical mutation method such as UV irradiation or radiationirradiation, as well as a chemical mutation method which utilizes amutation agent such as N-nitrosoguanidine, ethyl methane sulfonate,nitrite, methyl methane sulfonate, an acridine dye, benzopyrene, anddimethyl sulfate.

The method of screening a microorganism for producing scyllo-inososefrom the Acetobacter sp. which has been subjected to the mutationtreatment is exemplified hereinafter. However, the screening method isnot limited to the following method as long as the screening method isperformed based on the NAD⁺-independent myo-inositol 2-dehydrogenaseactivity.

The AB10253 strain which has been subjected to a mutating treatment isspread on a agar medium containing myo-inositol and nutrients such that10 to 300 colonies, preferably 100 to 150 colonies are formed per a dishhaving a diameter of 9 cm. Here, a carbon source, a nitrogen source, andother nutrients to be added as nutrients to the medium, which are knownto be conventionally used for general microorganism culture, can beused. Examples of the carbon source include glucose, sucrose, maltose,and a starch. Preferably, the concentration of the carbon source to beadded is 0.1% to 20%, more preferably 0.3% to 5%. Examples of thenitrogen source include peptone, a yeast extract, casamino acid,ammonium sulfate, ammonium chloride, ammonium nitrate, urea, and meatextract. Preferably, the concentration of the nitrogen source to beadded is 0.01% to 5.0%, preferably 0.5% to 2.0%.

In addition, if necessary, inorganic salts each capable of forming anion of sodium, potassium, calcium, magnesium, cobalt, manganese, zinc,iron, copper, molybdenum, phosphoric acid, sulfuric acid, or the likeare suitably added to the medium. The expression of the enzyme of thepresent invention is efficiently induced by culturing in the culturesolution adjusted to pH of 3 to 10, preferably pH of 5 to 7.

The culture may be performed until adequate numbers of colony areformed, and colonies are formed in about 3 days. The culture temperatureis preferably 25 to 30° C., preferably 27° C., which is the optimumtemperature for the growth of the microorganism.

Colonies are isolated and cultured and the NAD⁺-independent myo-inositol2-dehydrogenase activity of each colony is determined, to allowselection of strains each exhibiting a strong activity. Furthermore, asdescribed hereinafter, the strains can be efficiently selected on anagar medium using a 9 cm-dish.

After the culture, 10 ml of an agar medium for analysis is slowly pouredonto the colonies formed on the 9 cm-dish. The agar medium for analysisis a viscous solution prepared by: adding an agar to be 0.5% into acomposition consisting of 100 mM phosphate buffer containing 1%myo-inositol and 0.4% oxidized DCIP; cooling the mixture to 36° C. afterthe agar had been dissolved, so that the agar does not solidify. Theagar medium for analysis as prepared above is applied is slowly cooledto 27° C., and solidified such that the agar medium is piled on thecolonies which had been formed on the 9 cm-dish.

After the treatment, the dish was incubated at 27° C. As a result, bluecolor of the oxidized DCIP spread on the entire agar medium is observed,and then gradually changes to transparent only around the colonies,owing to the degree of the NAD⁺-independent myo-inositol 2-dehydrogenaseactivity. At this time, a colony of which its surrounding changed totransparent faster than others is transferred to a fresh medium. Thus, astrain having a high NAD⁺-independent myo-inositol 2-dehydrogenaseactivity can be obtained.

Meanwhile, a strain capable of converting myo-inositol intoscyllo-inosose using oxygen as an electron acceptor can be bred by:further subjecting the scyllo-inosose-producing microorganism obtainedas described above to a mutation treatment; and screening among theresultant strains using aerobic respiration ability as an index. Here,the term “aerobic respiration ability” means an ability to grow wellunder a low-oxygen condition. The term “low-oxygen condition” means acondition where the oxygen concentration is, for example, 3% or less.The Acetobacter sp. AB10253 strain is a strictly aerobic bacterium,therefore, a strain having high oxygen respiration ability can beobtained by transferring a colony that grows well under a low-oxygencondition into a fresh medium.

Moreover, the above-described screening method can be applied to anatural microorganism. That is, another screening method of the presentinvention is a screening method, comprising isolating a microorganismfrom a natural sample containing microorganisms; and selecting amicroorganism based on the NAD⁺-independent myo-inositol 2-dehydrogenaseactivity from the isolated microorganisms. Here, an example of thesample containing natural microorganisms includes soil. An example ofthe method of isolating a microorganism from a natural sample includes amethod comprising: applying a suspension of the natural sample or adiluent thereof onto an agar medium; and allowing the microorganism inthe natural sample to grow as an independent colony on the agar medium.The same operation as that of the case of using the Acetobacter sp.AB10253 strain can be applied to a method of screening a microorganismfor the production of scyllo-inosose from the isolated microorganisms,except that the pH of the medium is adjusted to 3 to 4, preferably 3.5.

<2-4> Method of Producing Scyllo-inosose

The present invention also relates to a method of producingscyllo-inosose, comprising reacting myo-inositol with NAD⁺-independentmyo-inositol 2-dehydrogenase or a strain having a high NAD⁺-independentmyo-inositol 2-dehydrogenase activity (a microorganism for producingscyllo-inosose).

(i) Method of Producing Scyllo-inosose Using NAD⁺-IndependentMyo-inositol 2-dehydrogenase

The “method of producing scyllo-inosose using NAD⁺-independentmyo-inositol 2-dehydrogenase” of the present invention is a producingmethod comprising: producing scyllo-inosose by reacting NAD⁺-independentmyo-inositol 2-dehydrogenase with myo-inositol in a solution containingthe myo-inositol and an electron acceptor; and separating and isolatingthe produced scyllo-inosose from the solution. Here, theNAD⁺-independent myo-inositol 2-dehydrogenase that is obtained by thedescribed method can be used. The degree of the purification of theenzyme of the present invention may be any degree as long as the enzymehas the activity to produce scyllo-inositol from myo-inositol.

Myo-inositol as a substrate is used at the concentration of 0.1% to 20%,preferably 5% to 10%. For the enzyme solution to be used in the reactionof the present invention, the above-described crude enzyme solution oran enzyme solution that is highly purified can be used. An alkalinesolution or acidic solution can be appropriately added or an appropriatebuffer solution can be used to preferably maintain the pH of thereaction to be 5.0 while monitoring the pH. A buffer having a bufferingability around pH of 5.0 can be used without particular limitation, anda phosphate buffer is preferably used.

In the producing method that utilizes the enzyme, an electron acceptingsubstance has to be added to the reaction solution. Here, examples ofthe electron accepting substance include an oxidized DCIP, phenazinemethosulfate (PMS), methylene blue, and Fe³⁺ ion, and a combinationthereof can be used, however, an oxidized DCIP is preferably used. Theamount of the electron accepting substance to be added is 1 mol withrespect to 1 mol of myo-inositol. The electron accepting substance canbe appropriately added with respect to a corresponding mol number ofmyo-inositol. When the concentration of those electron acceptingsubstances increases as the reaction proceeds, reduced electronaccepting substances may precipitate. In this case, the precipitates canbe removed by means of an operation such as centrifugal separation orfiltration. The reaction of the present invention may be a nonuniformsystem according to the solubility of the electron accepting substance,and therefore the reaction is preferably carried out under stirring.

The reaction temperature of the reaction of the present invention is notlimited as long as the enzyme does not lose its activity, however thereaction can be preferably carried out at a temperature of 20° C. to 40°C. The reaction time is preferably 1 to 72 hours, more preferably 8 to12 hours. The produced scyllo-inosose can be separated and purified by arecrystallization method or the like.

(ii) Method of Producing Scyllo-inosose Using a Microorganism

The present invention also relates to a method of producingscyllo-inosose using a microorganism, comprising: producingscyllo-inosose from myo-inositol by culturing a microorganism for theproduction of scyllo-inosose that is obtained by the screening method ona medium containing myo-inositol; and separating and purifying theproduced scyllo-inosose from the medium.

Composition of the liquid medium to be used herein is not particularlylimited as long as the microorganism can produce scyllo-inosose frommyo-inositol. For example, the liquid medium can contain myo-inositol asa material to be converted into scyllo-inositol, in addition to carbonsources, nitrogen sources, organic nutrients, inorganic salts, and thelike. Both a synthetic medium and a natural medium can be used.Specifically, a liquid medium contains: preferably 0.1% to 40%, morepreferably 10% to 30% of myo-inositol; preferably 0.1% to 20%, morepreferably 0.3% to 5% of glycerol, sucrose, maltose, or a starch as acarbon source; and 0.01% to 5.0%, preferably 0.5% to 2.0% of a yeastextract, peptone, casamino acid, ammonium sulfate, ammonium chloride,ammonium nitrate, urea, or the like as a nitrogen source.

In addition, if necessary, inorganic salts each capable of forming anion of sodium, potassium, calcium, magnesium, cobalt, manganese, zinc,iron, copper, molybdenum, phosphoric acid, sulfuric acid, or the likecan be suitably added into the medium. Scyllo-inositol is efficientlyproduced by culturing in the culture solution adjusted to pH of 4 to 10,preferably 5 to 9.

Culture conditions vary depending on a kind of the strain or medium.However, a culture temperature is preferably 12 to 35° C., morepreferably 20 to 27° C. Culture may be preferably performed aerobicallyby, for example, shaking the liquid medium or aerating air or an oxygengas into the liquid medium. A culture period may be preferably untilwhen the myo-inositol in the culture solution is completely consumed andthe accumulation of the scyllo-inositol becomes the maximum, and isgenerally 1 to 10 days, preferably 3 to 8 days.

A general method of separating and purifying a general aqueous neutralsubstance can be applied for the method of separating and purifying atarget substance from the culture solution. For example, a supernatantof the culture solution is treated with activated carbon, an ionexchange resin, or the like after cells had been removed from theculture solution, to thereby allow most impurities other thanscyllo-inosose to be removed. However, it is preferable not to use OH⁻type of a strong basic anion exchange resin because it chemicallychanges scyllo-inosose. After that, the target substance can beseparated by using a method such as recrystallization.

A specific method of separating and purifying scyllo-inosose isexemplified hereinbelow. However, the separating and purifying method isnot limited thereto. First, a supernatant of the culture solution havingaccumulated scyllo-inosose is passed through a column filled with astrong acidic cation exchange resin such as Duolite® C-20 (H⁺ type)(manufactured by Sumitomo Chemical Co., Ltd.) to remove undesirablecomponents. After a flow-through solution is collected, deionized wateris passed through the column to wash the column, to thereby collect awash solution. The obtained flow-through solution and the wash solutionare combined together. Thus obtained solution is passed through a columnfilled with a weak basic anion exchange resin such as Duolite® A368S(free base form). After a flow-through solution is collected, deionizedwater is passed through the column to wash the column, to therebycollect a wash solution. The obtained flow-through solution and the washsolution are combined together, to obtain a solution containingscyllo-inosose but almost no other impurities. The solution wasconcentrated to thereby obtain a concentrated solution ofscyllo-inosose. The concentrated solution was added with an appropriateamount of ethanol and left overnight at room temperature or a lowtemperature, to thereby allow a pure scyllo-inosose to be crystallized.

<2-5> Method of Producing Scyllo-inositol

The present invention also relates to a method of producingscyllo-inositol, comprising: producing scyllo-inosose from myo-inositolusing a NAD⁺-independent myo-inositol 2-dehydrogenase or a strain thatshows high activity of the enzyme; and obtaining scyllo-inositol byreducing the obtained scyllo-inosose.

In the producing method, the step of producing scyllo-inosose frommyo-inositol using a NAD⁺-independent myo-inositol 2-dehydrogenase or astrain that shows high activity of the enzyme (microorganism for theproduction of scyllo-inosose) can be performed by a method describedabove. The scyllo-inosose obtained from the step may be used for thereducing step after being isolated and purified, or may be used for thereducing step without being isolated and purified. In the case ofproducing scyllo-inosose using a microorganism for the production ofscyllo-inosose, a cultured filtrate obtained by separating only cellswithout isolating scyllo-inosose from a culture solution in whichscyllo-inosose has been produced and accumulated may be used for thereducing step.

Examples of a reducing agent capable of reducing scyllo-inosose intoscyllo-inositol in a reaction solution system include, but not limitedto, sodium borohydride, lithium borohydride, potassium borohydride,sodium trimethoxy borohydride, and cyanated sodium borohydride.Reduction of scyllo-inosose using those reducing agents results inproduction of scyllo-inositol and myo-inositol. Production ratiosthereof vary depending on a reaction temperature or kinds of reducingreagents, however, generally a mixture consisting of scyllo-inositol andmyo-inositol in a ratio of about 4:6 can be obtained. Thus,scyllo-inositol has to be separated and purified from the mixture.

Furthermore, the reduction of scyllo-inosose into scyllo-inositol may beperformed using a novel scyllo-inositol dehydrogenase provided by thepresent invention as described hereinbelow.

In order to separate and purify scyllo-inositol from a reduced reactionsolution, a general method of isolating and purifying a normal aqueousneutral substance can be applied. For example, at first, a reactionsolution is treated with activated carbon, an ion exchange resin, or thelike, to thereby obtain an aqueous solution containing scyllo-inositoland myo-inositol but almost no other impurities. In order to obtain onlyscyllo-inositol from the aqueous solution, it is effective to utilizethe difference in solubility to water. That is, a method of obtainingscyllo-inositol comprising: concentrating the aqueous solution; andallowing the scyllo-inositol having low solubility to water to beprecipitated as a solid can be used.

Furthermore, as described hereinbelow, scyllo-inositol may be separatedand purified by a method comprising: adding boric acid and NaCl to theobtained scyllo-inositol-containing solution to thereby forming ascyllo-inositol/boric acid complex; filtrating and separating thecomplex; allowing the boric acid to be released by means of the acid;and allowing the scyllo-inositol to be crystallized by adding an organicsolvent such as methanol.

3. Scyllo-inositol Dehydrogenase and a Method of ProducingScyllo-inositol Using the Same

Other embodiment of the present invention relates to a novel enzymescyllo-inositol dehydrogenase and a method of producing scyllo-inositolusing the enzyme.

<Scyllo-inositol Dehydrogenase>

As represented in the following reaction formula, scyllo-inositoldehydrogenase of the present invention catalyzes an oxidation-reductionreaction between scyllo-inositol and scyllo-inosose, andstereospecifically reduces scyllo-inosose into scyllo-inositol in thepresence of NADH or NADPH.

The origin of such a scyllo-inositol dehydrogenase is not limited aslong as it has the above-described activity. However, scyllo-inositoldehydrogenase is preferably derived from microorganisms and particularlypreferably derived from Escherichia coli, the genus Acetobacter, thegenus Bacillus, the genus Agrobacterium, the genus Xanthomonas, or thelike. It is particularly preferably derived from Escherichia coli K-12strain ATCC10798, Acetobacter sp. AB10281 strain FERM BP-10119, Bacillussubtilis 168 strain ATCC23857, Agrobacterium tumefacience C5-8 strainATCC33970, Agrobacterium sp. AB10121 strain FERM P-17383, or Xanthomonascampestris pv. campestris ATCC33913.

The scyllo-inositol dehydrogenase of the present invention includes ascyllo-inositol dehydrogenase having the following physiologicalproperties.

Reaction: as shown by the following reaction formula, it catalyzes theoxidation-reduction reaction between scyllo-inositol and scyllo-inososeand stereospecifically reduces scyllo-inosose into scyllo-inositol inthe presence of NADH or NADPH.

The method of determining the activity of scyllo-inositol dehydrogenasecan be either of a method of determining the reducing activity or amethod of determining oxidizing activity. However, the method ofdetermining the reducing activity is preferable, because accuracy is lowin the determination of the oxidizing activity due to the activityderived from co-existing myo-inositol 2-dehydrogenase and a faintactivity of the oxidizing activity itself.

The determination of the reducing activity is achieved by determining adecrease in absorption at 340 nm of NADH or NADPH under a condition inwhich scyllo-inositol is used as a substrate and coexists with NADH orNADPH. Furthermore, the determination can be achieved by determiningwhether a product in a solution after completion of the reaction isscyllo-inositol or myo-inositol by means of an analyzing apparatus suchas HPLC or GLC.

Furthermore, the scyllo-inositol dehydrogenase of the present inventionpreferably has the following physiological properties.

Molecular weight and association property: 38 to 46 k Dalton, thescyllo-inositol dehydrogenase forms a dimer or a trimer.

The molecular weight can be calculated as an estimated molecular weightof the enzyme from a result of sodium dodecyl sulfate-polyacrylamideelectrophoresis (SDS-PAGE) or the like or based on the full length ofthe DNA. The association property can be determined by: measuring theactivity of a faction fractionated using a gel filtration column(2000SWXL, manufactured by Tosoh Corporation); calculating the molecularweight of a corresponding molecular weight; and making a value obtainedby dividing the molecular weight by the molecular weight of the enzymeinto an integer.

Further, the scyllo-inositol dehydrogenase of the present inventionpreferably has the following physiological properties.

Coenzyme: NAD⁺ or NADP⁺, or NADH or NADPH is used as a coenzyme.

Selectivity of the coenzyme can be confirmed by: mixing 5 μl of areaction solution (200 mM Tris buffer of pH 8.0, 2% of NADPH or NADH,and 1% of scyllo-inosose) and 5 μl of an enzyme solution; allowing themixture to react at 36° C. for 30 minutes; adding 500 μl of waterimmediately after the completion of the reaction; determining theabsorbance at 340 nm; and determining a decrease in the absorbance at340 nm with respect to a blank value of a test solution including waterinstead of the enzyme solution. The values indicated by “%” eachrepresent a percentage of weight/volume (w/v), and hereinafter, it alsohas the same meaning when a concentration is indicated by “%”.

Further, a coenzyme-relative activity can be confirmed using theabove-described determination method. The enzyme of the presentinvention can be divided into groups each having NADPH:NADH ratio of100:1 to 100:10, 100:10 to 100:30, 100:30 to 100:60, and 100:60 to100:120 according to the coenzyme-relative activities.

Furthermore, the scyllo-inositol dehydrogenase of the present inventionpreferably has the following physiological properties.

Activation by heavy metals: it is activated in the presence of Co²⁺ ion,or may be activated in the presence of Mn²⁺, Zn²⁺, or Ca²⁺ ion.

Inhibition by heavy metals: it is inhibited in the presence of Sn²⁺ ion,or may be inhibited in the presence of Zn²⁺ ion.

Effects by the heavy metals can be confirmed by: mixing 5 μl of areaction solution (200 mM Tris buffer of pH 8.0 containing 2% of NADPH,1% of scyllo-inosose, and 2 mM metal salt) and 5 μl of an enzymesolution; allowing the mixture to react at 36° C. for 30 minutes; adding500 μl of water immediately after the completion of the reaction;determining the absorbance at 340 nm; and determining a decrease in theabsorbance at 340 nm with respect to a blank value of a test solutioncontaining water instead of the enzyme solution. The term “activated”means the case where the enzymatic activity of a fraction added with 1mM of a heavy metal is 105% or more, preferably 120% or more, withrespect to the enzymatic activity of a fraction added with no heavymetal as 100%. On the other hand, the term “inhibited” means the casewhere the enzymatic activity of the fraction added with 1 mM of a heavymetal is 95% or less, preferably 70% or less, with respect to theenzymatic activity of the fraction added with no heavy metal as 100%.

Further, the scyllo-inositol dehydrogenase of the present inventionpreferably has the following physiological properties.

Optimum pH: the enzyme of the present invention has an activity at pH of5 to 9.

The optimum pH can be confirmed by: mixing 5 μl of a reaction solution(200 mM Tris buffer of pH 5.0 to 9.0 containing 2% of NADPH, and 1% ofscyllo-inosose) and 5 μl of an enzyme solution; allowing the mixture toreact at 36° C. for 30 minutes; adding 500 μl of water immediately afterthe completion of the reaction; determining the absorbance at 340 nm;and determining a decrease in the absorbance at 340 nm with respect to ablank value of a test solution containing water instead of the enzymesolution. The term “optimum pH” means a pH at which the enzyme has 90%or more of the maximum activity. The enzyme of the present invention canbe divided depending on, for example, a range of the optimum pH intogroups each having: an optimum pH in an acidic region (optimum pH is pHof 5.5 to 6.5); an optimum pH in a neutral region (optimum pH is pH of6.5 to 7.5 or 6.5 to 8.5); and an optimum pH in an alkaline region(optimum pH is pH of 7.0 to 8.5 or 7.5 to 9.0).

Further, the scyllo-inositol dehydrogenase of the present inventionpreferably has the following physiological properties.

Thermal stability: the scyllo-inositol dehydrogenase of the presentinvention is stable at up to 60° C.

The thermal stability can be confirmed by: treating an enzyme solutionat a predetermined temperature for 10 minutes followed by cooling it;mixing the enzyme solution and 5 μl of a reaction solution (200 mM Trisbuffer of pH 5.0 to 9.0 containing 2% of NADPH, and 1% ofscyllo-inosose); allowing the mixture to react at 36° C. for 30 minutes;adding 500 μl of water immediately after the completion of the reaction;determining the absorbance at 340 nm; and determining a decrease in theabsorbance at 340 nm with respect to a blank value of a test solutioncontaining water instead of the enzyme solution. The term “thermallystable” means a case where the above-described enzyme which washeat-treated still has 90% or more of its activity with respect to theactivity of a fraction which was treated at 20° C. for 10 minutes as100%.

Further, the scyllo-inositol dehydrogenase of the present inventionpreferably has the following physiological properties.

Km value with respect to scyllo-inosose: Km value with respect toscyllo-inosose is 2 to 13 mM.

The Km value with respect to scyllo-inosose can be confirmed by: mixing5 μl of a reaction solution (200 mM Tris buffer of pH 8.0 containing 2%of NADPH, and 0.001 to 2.5% of scyllo-inosose) and 5 μl of an enzymesolution; allowing the mixture to react at 36° C. for 30 minutes; adding500 μl of water immediately after the completion of the reaction;determining the absorbance at 340 nm; and determining a decrease in theabsorbance at 340 nm with respect to a blank value of a test solutioncontaining water instead of the enzyme solution. The determination iscarried out such that the Km value is calculated after reciprocal plotaccording to the general method. The enzyme of the present invention canbe divided into groups, for example, each having a Km value with respectto scyllo-inosose of less than 4 mM, not less than 4 mM and less than 10mM, and not less than 10 mM and less than 13 mM, respectively.

Further, the scyllo-inositol dehydrogenase of the present inventionpreferably has the following physiological properties.

Substrate specificity: Examples of a substrate having a relativeactivity of 70% or more include scyllo-inositol (SI), myo-inositol (MI),D-chiro-inositol (DCI), epi-inositol (EI), and L-chiro-inositol (LCI).Examples of a substrate having a relative activity of 20% or more andless than 70% include L-chiro-inositol (LCI), epi-inositol (EI),muco-inositol (MuI), myo-inositol (MI), D-chiro-inositol (DCI),allo-inositol (AI), and neo-inositol (NI). Examples of a substratehaving a relative activity of less than 20% include allo-inositol (AI),neo-inositol (M), D-chiro-inositol (DCI), L-chiro-inositol (LCI),epi-inositol (EI), and muco-inositol (MuI).

The substrate specificity can be confirmed by determining a relativeactivity with respect to the reactivity to scyllo-inositol based on theoxidizing activity. Examples of an inositol isomer includescyllo-inositol (SI), myo-inositol (MI), D-chiro-inositol (DCI),L-chiro-inositol (LCI), epi-inositol (EI), muco-inositol (MuI),allo-inositol (AI), and neo-inositol (NI). The substrate specificity ofscyllo-inositol dehydrogenase of the present invention can be shown asdivided sections each having a relative activity of not less than 70%,less than 70% and not less than 20%, and less than 20%.

The determination method can be performed by: mixing 50 μl of a reactionsolution (200 mM Tris buffer having of pH 8.0 containing 1% of thevarious inositol isomers (only neo-inositol is 0.4%), 0.002% of NADP⁺,0.002% of diaphorase, and 0.01% of nitrotetrazolium blue) and 50 μl ofan enzyme solution; and determining an increase in the absorbance at 545nm every 3 minutes at 25° C. using a microplate reader.

Those physiological properties preferably have a combination of any ofthe physiological properties.

<Production and Purification of Scyllo-inositol Dehydrogenase>

Examples of the microorganism to be used for the production ofscyllo-inositol dehydrogenase include, but not limited as long as themicroorganism has the ability to produce the enzyme, Escherichia coliK-12 strain ATCC10798 (hereinafter, also referred to as Escherichia coliK-12 strain), Acetobacter sp. AB10281 strain FERM BP-10119 (hereinafter,also referred to as AB10281 strain), Bacillus subtilis 168 strainATCC23857 (hereinafter, also referred to as Bacillus sub. 168 strain orB. sub. 168 strain), Agrobacterium tumefacience C58 strain ATCC33970(hereinafter, also referred to as A. tume. C58 stain), Agrobacterium sp.AB10121 strain FERM P-17383 (hereinafter, also referred to as an AB10121strain), and Xanthomonas campestris pv. campestris strain ATCC33913(hereinafter, also referred to as X. camp.).

For producing scyllo-inositol dehydrogenase, conventionally known commonmedia for a microorganism can be used as a medium for culturing thosemicroorganisms. For example, the composition of a medium to be used forculture of Acetobacter sp. AB10281 strain FERM BP-10119, Bacillussubtilis 168 strain ATCC23857, Agrobacterium tumefacience C58 strainATCC33970, Acetobacter sp. AB10121 strain FERM P-17383, or Xanthomonascampestris pv. campestris strain ATCC33913 is not particularly limitedas long as the object can be achieved. The medium may be a mediumcontaining myo-inositol which is a raw material to be converted intoscyllo-inositol, in addition to carbon sources, nitrogen sources,organic nutrients, inorganic salts, and the like. Both a syntheticmedium and a natural medium can be used. The medium is preferably addedwith 0.1% to 40%, more preferably 10% to 30% of myo-inositol; 0.1% to20%, more preferably 0.3% to 5% of glycerol, sucrose, maltose, or starchas a carbon source; and 0.01% to 5.0%, preferably 0.5% to 2.0% of ayeast extract, peptone, casamino acid, ammonium sulfate, ammoniumchloride, ammonium nitrate, urea, or the like as a nitrogen source. Inaddition, if necessary, inorganic salts each capable of forming an ionof sodium, potassium, calcium, magnesium, cobalt, manganese, zinc, iron,copper, molybdenum, phosphoric acid, sulfuric acid, or the like can beadded to the medium. A hydrogen-ion concentration in the culturesolution does not particularly need to be adjusted. However, cellscontaining scyllo-inositol dehydrogenase are efficiently obtained in theculture solution adjusted to preferably pH of 4 to 10, more preferablypH of 5 to 9.

Culture conditions vary depending on a kind of the medium. However, aculture temperature is 12 to 38° C., preferably 20 to 27° C. Culture maybe performed aerobically by, for example, shaking the liquid medium oraerating air or an oxygen gas into the liquid medium. A culture periodmay be until when the accumulation of the scyllo-inositol dehydrogenasebecomes maximum or an amount required to obtain an adequate activity,and is generally 1 to 10 days, preferably 3 to 8 days.

Meanwhile, the composition of a medium to be used for culturingEscherichia coli K-12 strain ATCC 10798 is not particular limited aslong as it accomplishes the object. The medium may be a mediumcontaining carbon sources, nitrogen sources, organic nutrients,inorganic salts, and the like. Both a synthetic medium and a naturalmedium can be used. Examples of the medium include LB medium, TB medium,and YT medium. In addition, 0.05 to 1%, preferably 0.5% of sorbose ispreferably added to the medium, as a substance which increases thespecific activity of scyllo-inositol dehydrogenase of the Escherichiacoli K-12 ATCC10798 strain about three times larger. Culture conditionsvary depending on a kind of the medium. However, a culture temperatureis 28 to 38° C., preferably 36° C. Culture may be performed aerobicallyby, for example, shaking the liquid medium or aerating air or an oxygengas into the liquid medium. A culture period may be until when theaccumulation of the scyllo-inositol dehydrogenase becomes a maximum oran amount required to obtain an adequate activity, and is generally 1 to3 days, preferably 1 day.

Scyllo-inositol dehydrogenase can be obtained by separating andpurifying the enzyme from thus cultured cells. The separation andpurification of the enzyme can be performed in the same manner as ageneral purification method for a protein. The separation andpurification method will be specifically described hereinbelow, however,it is not limited thereto.

First, in order to collect the cells after culture, a method such ascentrifugal separation or membrane concentration can be used. Ifnecessary, at this point, the cells can be washed by being suspended toan appropriate solution and being collected again using a method such ascentrifugal separation or membrane concentration. Thus obtained cellsare disrupted by means of a physical method such as use of an auto-millor ultrasonic, to thereby extract the enzyme of the present inventionwhich presents in the cells.

The cell-lysis solution containing the disrupted cells is divided intosoluble components and insoluble components using a method such ascentrifugal separation or membrane concentration. After that, the enzymeof interest can be purified from the soluble components in accordancewith a general procedure for enzyme purification. That is, a columnoperation using an affinity column such as Blue-Toyopearl (manufacturedby Tosoh Corporation), an ion exchange column typified by a DEAE columnor a CM column, a gel-filtration column, or a hydroxyapatite column, aswell as a batch-wise operation method such as an ammonium sulfatefractionation method or isoelectrite precipitation method can be used.

The method of determining the scyllo-inositol dehydrogenase activity canbe either of the method of determining the reducing activity or themethod of determining the oxidizing activity. However, the method ofdetermining the reducing activity is preferable, because accuracy is lowin the determination of the oxidizing activity due to the activity ofco-existing myo-inositol 2-dehydrogenase and activity of the oxidizingactivity itself is low. The determination of the reducing activity isachieved by determining a decrease in absorption of NADH or NADPH at 340nm under a condition in which scyllo-inosose is used as a substrate andcoexists with NADH or NADPH. Furthermore, the determination can beachieved by determining whether a product in a solution after completionof the reaction is scyllo-inositol or myo-inositol by means of ananalyzing apparatus such as HPLC or GLC.

The degree of purification of the enzyme can be confirmed byelectrophoresis using Native-PAGE, sodium dodecyl sulfate (SDS)-PAGE, orthe like. In addition, the corresponding protein can be highly purifiedby trans-blotting the enzyme to a protein adsorptive membrane such as aPVDF membrane.

Specific examples of the scyllo-inositol dehydrogenase of the presentinvention include the following proteins (a) and (b).

(a) A protein consisting of an amino acid sequence of SEQ ID NO: 2, 4,6, 8, 10, 12, 14, or 28.

(b) A protein consisting of an amino acid sequence of SEQ ID NO: 2, 4,6, 8, 10, 12, 14, or 28, including substitution, deletion, insertion,and/or addition of one or plural of amino acids; and catalyzing theoxidation-reduction reaction between scyllo-inositol and scyllo-inosose;and having an enzymatic activity to stereospecifically reducescyllo-inosose into scyllo-inositol in the presence of NADH or NADPH.

Of those, a scyllo-inositol dehydrogenase having the amino acid sequenceof SEQ ID NO: 28 is a novel protein provided by the present invention.

The term “protein which has an amino acid sequence includingsubstitution, deletion, insertion, and/or addition of one or plural ofamino acids; catalyzes the oxidation-reduction reaction betweenscyllo-inositol and scyllo-inosose; and has an enzymatic activity tostereospecifically reduce scyllo-inosose into scyllo-inositol in thepresence of NADH or NADPH” means that the protein may have substitution,deletion, insertion, and/or addition of one or plural of amino acidresidues which does not substantially inhibit the enzymatic activity tocatalyze the oxidation-reduction reaction between scyllo-inositol andscyllo-inosose and to stereospecifically reduce the scyllo-inosose intothe scyllo-inositol in the presence of NADH or NADPH.

That is, a naturally-occurring protein may have variations such assubstitution, deletion, insertion, and/or addition of amino acidresidues in an amino acid sequence owing to polymorphism and variationof a DNA encoding the protein, as well as a modification reaction or thelike in a cell after and during purification of the protein. However,some of the naturally-occurring proteins which may have variations areknown to have physiological and biological activities which aresubstantially the same as those of a protein having no variation. Asdescribed above, a protein having slight differences in the structurebut has no significant difference in the function is included in theprotein of the present invention. A protein made by artificiallyintroducing the above-described variations into the amino acid sequenceis also included, and further various mutants can be produced in thiscase. Furthermore, a certain kind of a protein is known to have apeptide region not essential for the activity. Examples of such apeptide region include a signal peptide present in a protein which isextracellularly secreted and a pro-sequence existing in a proteaseprecursor or the like. Most of those regions are removed aftertranslation or upon conversion into a mature protein. Such proteins havedifferent primary structures but are proteins finally having the samefunction, therefore they are included in the scyllo-inositoldehydrogenase of the present invention.

The term “plural of amino acids” as used herein indicates the number ofamino acids which may be mutated so long as the activity of the enzymeof the present invention is not lost. For example, in the case of apolypeptide consisting of 400 amino acid residues, the number is about 2to 20, preferably 2 to 10, more preferably 2 to 3. Furthermore, aprotein having a homology of not less than 80%, more preferably not lessthan 90%, still more preferably not less than 95%, or particularlypreferably not less than 98% to the protein of the present invention isincluded in the scyllo-inositol dehydrogenase of the present invention.

Further, examples of the scyllo-inositol dehydrogenase of the presentinvention include those each encoded by the following DNAs.

(a) A DNA comprising a coding region of the nucleotide sequence of SEQID NO: 1, 3, 5, 7, 9, 11, 13, or 27.

(b) A DNA which: hybridizes under stringent conditions with a DNA havingthe nucleotide sequence of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, or 27 or anucleotide sequence complementary to the nucleotide sequence; andencodes a protein which catalyzes the oxidation-reduction reactionbetween scyllo-inositol and scyllo-inosose and has an enzymatic activityto stereospecifically reduce scyllo-inosose into scyllo-inositol in thepresence of NADH or NADPH.

Of those, a DNA having the nucleotide sequence of SEQ ID NO: 27 is anovel DNA encoding scyllo-inositol dehydrogenase provided by the presentinvention.

The term “stringent conditions” used herein mean conditions where aso-called specific hybrid is formed and nonspecific hybrid is not formed(see, Sambrook, J. et al., Molecular Cloning A Laboratory Manual, SecondEdition, Cold Spring Harbor Laboratory Press (1989), and the like). Aspecific example of the “stringent condition” includes a condition inwhich: hybridization is performed in a solution containing 50%formamide, 4×SSC, 50 mM HEPES (pH of 7.0), 10×Denhardt's solution, and100 g/ml of a salmon sperm DNA at 42° C.; and then washing is performedwith 2×SSC, 0.1% SDS solution at room temperature and 0.1×SSC, 0.1% SDSsolution below 50° C. In other words, the examples include a conditionwhere DNAs having homology of preferably not less than 80% or more, morepreferably not less than 90%, still more preferably not less than 95%,and particularly preferably not less than 98% specifically hybridize.That is, a DNA having homology of not less than 80%, more preferably notless than 90%, still more preferably not less than 95%, and particularlypreferably not less than 98% to the nucleotide sequence of SEQ ID NO: of1, 3, 5, 7, 9, 11, 13, or 27 is included in the DNA encoding thescyllo-inositol dehydrogenase of the present invention.

<Method of Producing Scyllo-inositol Using Scyllo-inositolDehydrogenase>

Furthermore, the present invention relates to a method of producingscyllo-inositol, comprising: subjecting inexpensive myo-inositol as asubstrate to enzyme conversion via scyllo-inosose into scyllo-inositolin the presence of NADH or NADPH in a solution in which scyllo-inositoldehydrogenase and myo-inositol 2-dehydrogenase coexist (see, FIG. 1).

The scyllo-inositol dehydrogenase to be used in the producing method maybe an enzyme produced by purification of the above-described enzyme ormay be a recombinant enzyme produced by genetic engineering using a DNAencoding scyllo-inositol dehydrogenase.

The DNA encoding scyllo-inositol dehydrogenase can be obtained, forexample, by amplifying the DNA having the nucleotide sequence of SEQ IDNO: 1, 3, 5, 7, 9, 11, 13, or 27 by means of a polymerase chain reaction(PCR) using the whole genome extracted from a microorganism as atemplate.

Further, the DNA encoding scyllo-inositol dehydrogenase can be obtainedby isolating a homologous DNA searched based on a homology. Here, it ishighly likely that a series of genes having high homology which arereferred to as ydgJ gene is a DNA encoding scyllo-inositoldehydrogenase.

Thus obtained DNA is inserted into a plasmid vector. A plasmid vector tobe used at this time is preferably an expression plasmid vector having amulti-cloning site, however, other plasmid vector which is capable ofexpressing an enzyme and has restriction enzyme sites can be used. Aplasmid can be constructed by: attaching an appropriate restrictionenzyme site on a terminal of a fragment; and ligating the site with thesame restriction enzyme site present on the plasmid vector.

Further, a promoter to be used for expression of the DNA which has beeninserted in the plasmid is not particularly limited as long as the DNAis expressed in a host microorganism. For example, a lac promoter, tacpromoter, or the like can be used.

Thus prepared recombinant plasmid vector can be introduced into a hostmicroorganism. A host microorganism to be used at this time is notparticularly limited as long as the recombinant plasmid vector is stableand autonomously replicable. A host microorganism which is generallyused for gene recombination such as a microorganism belonging to thegenus Escherichia or the genus Bacillus is preferably used, andEscherichia coli is more preferably used.

As a method of introducing the recombinant plasmid vector into the hostmicroorganism, a method of introducing a recombinant DNA in the presenceof an calcium ion or a competent cell method may be performed when thehost microorganism is a microorganism belonging to the genusEscherichia, and the competent cell method, a protoplast method, anelectroporation method, or a microinjection method can be used when thehost microorganism is a microorganism belonging to the genus Bacillus.Selection of the presence or absence of a desirable recombinant DNAintroduced into a host microorganism may be performed by: culturing thehost microorganisms of interest in a selection medium based on adrug-resistance marker contained in the recombinant plasmid vector; andselecting the host microorganisms which is grown.

Next, a medium to be used for the expression and induction of the enzymeis not particularly limited as long as the host microorganism stablygrows in the medium. Examples of such a medium include Nutrient Brothand L-Broth. Furthermore, depending on the kind of the plasmid vector,an inducer such as isopropyl thiogalactopyranoside (IPTG) may be addedin order to express a DNA in the medium or the inducer may be addedduring culture.

A host microorganism introduced with the recombinant plasmid vectorhaving thus prepared DNA is cultured to express the DNA of the presentinvention. The microorganism having the expressed enzyme of the presentinvention is subjected to centrifugal separation. The medium is removedand then the microorganism as a pellet is washed with water, followed bycentrifugal separation, to thereby obtain washed cells. The washed cellsare suspended in water or an appropriate solution, and the suspendedcells are disrupted. After disruption, centrifugal separation isperformed. Thus, a solution containing a recombinant enzyme derived fromthe DNA of the present invention in the supernatant can be obtained.

The cells expressing the recombinant enzyme can be subjected to areaction as a cell suspension obtained by adding the cells as they areto a reaction solution after being washed. However, a solutioncontaining the enzyme which had been present in the cells prepared bydisrupting the cells and extracting the enzyme is preferably used. Inaddition, the extract can be used after purification. The purificationincludes an ammonium sulfate fractionation treatment, or thepurification can be performed by column chromatography using a lineargradient of a salt concentration or a temperature treatment afteradsorption to an ion exchange resin. Furthermore, the enzyme of thepresent invention can be used as an immobilized enzyme or immobilizedcells. A general immobilizing method such as a gel embedding method oran ion exchange resin adsorbing method can be applied as an immobilizingmethod.

Meanwhile, an enzyme which oxidizes myo-inositol and producesscyllo-inositol in the presence of NAD⁺ or NADP⁺ is preferably used asmyo-inositol 2-dehydrogenase.

In the production method, a known NAD⁺ or NADP⁺-dependent myo-inositol2-dehydrogenase can be used. For example, a commercially availableenzyme, an enzyme produced by purification from cultured cells ofBacillus subtilis or Bacillus halodurans, or a recombinant enzymeexpressed by genetic engineering based on known gene sequence may beused.

Examples of the amino acid sequence of a known myo-inositol2-dehydrogenase are registered in the protein database of NationalCenter for Biotechnology Information (NCBI) under an accession numbersof 2636516, 17982589, 23464076, 10174936, 17742455, 50120397, 28853468,and 13422633. Also, information of nucleotide sequence encoding thoseamino acid sequences can be obtained by referring to CDS of theaccession numbers.

The cells expressing the recombinant enzyme can be subjected to areaction as a cell suspension by adding the cells as they are to areaction solution after being washed. However, a solution containing anenzyme which had been present in the cells prepared by disrupting thecells and extracting the enzyme is preferably used. In addition, theextract of the present invention can be used after purification. Thepurification includes an ammonium sulfate fractionation treatment, orthe purification can be performed by column chromatography using alinear gradient of a salt concentration or a temperature treatment afteradsorption to an ion exchange resin. Furthermore, the enzyme of thepresent invention can be used as an immobilized enzyme or immobilizedcells. A general immobilizing method such as a gel embedding method oran ion exchange resin adsorbing method can be applied to an immobilizingmethod.

When the recombinant enzyme is used, myo-inositol 2-dehydrogenase andthe enzyme of the present invention can be simultaneously expressed bymeans of genetic engineering, an enzyme solution prepared as such can beused.

The ratio of the activity (U) between the myo-inositol 2-dehydrogenaseand the scyllo-inositol dehydrogenase in the present reaction system isdefined by the number of units. When the scyllo-inosose is used as thesubstrate at 36° C., a rate at which 1 μmol of NADH or NADPH is consumedper 1 minute is defined as 1 U. In this case, it is desirable that theratio of the activity (U) of both enzymes would be 1:10 to 10:1,preferably 1:2 to 2:1.

The present reaction system requires the co-enzyme NAD⁺ or NADP⁺. It isshown that the coenzymes are recycled in the reaction solution since theco-enzymes are converted into NADH or NADPH, and NADH or NADPH are againconverted into NAD⁺ or NADP⁺. NAD⁺ or NADP⁺ and NADH or NADPH differs inpH stability in a solution. NAD⁺ or NADP⁺ is stable at pH of 8.0 orless, and NADH or NADPH is stable at pH of 8.0 or more. Therefore, thepH of the present reaction system is preferably maintained at about pH8.0.

Any one of NAD⁺, NADH, NADP⁺, and NADPH, or a mixture thereof can beused as the co-enzyme to be used in the present enzymatic reaction.However, NAD⁺ or NADP⁺ is desirable in view of the stability. It isdesirable to add it at a concentration of 0.0001 to 0.1%, preferably0.004 to 0.01%.

In addition, reaction rate of the present reaction significantlyincreases by adding scyllo-inosose, an intermediate of the presentreaction system, to the reaction solution. Thus, scyllo-inosose isdesirably added to become 0.01 to 3%, preferably 0.2 to 0.5% in thereaction solution.

Further, myo-inositol 2-dehydrogenase and scyllo-inositol dehydrogenasecan react at pH 8.0. Therefore, the pH of the solution for the enzymaticreaction is adjusted to pH ranging from 6.0 to 8.5, and they are reactedwith each other. However, it is desirable that the pH is preferably 7.7to 8.3, more preferably 8.0, in view of the stability of NAD⁺ or NADP⁺and scyllo-inosose. If necessary, a buffer may be added to the reactionto thereby maintain the pH during the reaction. The kind of the bufferto be added is not particularly limited, however, a buffer having abuffering ability at about pH 8.0 is desirable. More preferably, aphosphate buffer, Tris buffer, or the like is exemplified.

Furthermore, the myo-inositol 2-dehydrogenase is activated by Mg²⁺ ionand the scyllo-inositol dehydrogenase is activated by Co²⁺ ion. Thus,the addition of those metal ions increases the reaction rate. Therefore,it is desirable that Co salt and/or Mg salt is added to be 0.01 to 5.0mM, preferably 0.2 to 2.0 mM in the reaction solution. Any salt whichdissolves in water can be used as Co salt or Mg salt, and examplesthereof include salts of hydrochloride and sulfate.

It is desirable to use the myo-inositol, the substrate to be used in thepresent invention, at a concentration of 1 to 30%, preferably 5 to 22%in the reaction solution. As the reaction proceeds, scyllo-inositolwhich is oversaturated by more than 1.6% precipitates as a crystal,resulting in reduction of myo-inositol. Therefore, an amount ofmyo-inositol corresponding to the reduction is added to the reactionsolution to maintain the myo-inositol at a constant concentration, tothereby allow continuous reaction.

The reaction temperature is not particularly limited as long as thereaction proceeds. However, the reaction is preferably performed at 20to 50° C., more preferably 35 to 40° C. in view of the solubility of thesubstrate, the stability of NAD⁺ or NADP⁺, and the heat-stability of theenzyme. Stirring is necessary in a method in which the cells aresuspended since it is a heterogeneous reaction. Stirring is notnecessary when the extracted enzyme is used since it is a homogenoussolution, but stirring is preferably performed to make the temperatureuniform.

During the enzymatic reaction, scyllo-inositol which is a reactant canprecipitate as a crystalline scyllo-inositol when the concentration ofthe scyllo-inositol becomes equal to or larger than its solubility. Thereaction does not need to be terminated when a solid-liquid separationmethod such as filtration or decantation is used, and the reaction canbe continued by adding myo-inositol into a solution such as a filtratedsolution again.

When the enzymatic reaction needs to be terminated, the enzymaticreaction itself may be terminated. Methods including heating, changingpH, addition of a denaturing agent for a protein, or the like can beused. The heating is preferable in view of the subsequent step ofpurification of the scyllo-inositol. For example, the reaction solutioncan be heated to 70 to 120° C., preferably 80 to 90° C. for 10 to 20minutes.

In addition, the termination of the enzymatic reaction can be performedby separating the enzyme. The enzyme can be separated by passing thereaction solution through an ion exchange resin column. When animmobilized enzyme is used, the reaction solution is subjected tocentrifugal separation or a filtration operation to thereby collectingthe immobilized enzyme.

After the termination of the reaction or during the reaction,oversaturated scyllo-inositol precipitates as a crystal. The crystallinescyllo-inositol can be isolated by means of an operation such asfiltration or centrifugal separation. When the crystallinescyllo-inositol co-exists with cells and insoluble denatured proteins,the cells and insoluble denatured proteins can be removed by dissolvingthe crystalline scyllo-inositol in water followed by an operation suchas filtration or centrifugal separation.

A method of purifying thus obtained crystalline scyllo-inositol can beperformed as follows. The point to be noted at this stage is the removalof neo-inositol in the crystalline scyllo-inositol. Neo-inositol is asubstance generated such that: the fifth position of a part ofmyo-inositol is oxidized by scyllo-inositol dehydrogenase to generateneo-inosose; and the generated neo-inosose is reduced by themyo-inositol 2-dehydrogenase. In the present reaction system, a traceamount of the substance generates. Also, the neo-inositol is a substancehaving a low water-solubility of 0.5% and is apt to be precipitated as acrystal with the scyllo-inositol.

However, a scyllo-inositol solution obtained by dissolving thecrystalline scyllo-inositol in water again contains almost nomyo-inositol. Scyllo-inositol which contains a trace amount of theneo-inositol can be obtained by means of an operation such as filtrationor centrifugal separation for isolating a crystalline scyllo-inositol tobe generated by reconcentration of the scyllo-inositol solution. Inaddition, for higher purification, a recrystallized scyllo-inositol,which is recrystallized through concentration of the liquid after beingpurified by passing through a desalting column or activated carboncolumn, is isolated by using an operation such as filtration orcentrifugal separation, to yield a pure scyllo-inositol containing noneo-inositol.

A column employing an ion exchange resin is preferable for the desaltingcolumn. For the ion exchange resin to be used at this time, any one of astrong basic ion exchange resin and a weak basic ion exchange resin, ora combination thereof, or any one of a strong acidic ion exchange resinand a weak ion exchange resin, or a combination thereof can be used. Asa manner in which the ion exchange resin is reacted, a method comprisingpassing a solution through an ion exchange resin loaded in a column isoptimum. The solution may also be desalted by filtration after beingmixed in batch-wise manner and stirred with the ion exchange resin.

The activated carbon column is used for decolorization. A methodcomprising passing the solution through the activated carbon loaded in acolumn can be used. The solution may be decolorized by filtration afterbeing mixed in batch-wise manner and stirred with the activated carbon.

Next, after the termination of the enzymatic reaction, a method ofpurifying the soluble scyllo-inositol which dissolves in the reactionsolution can be performed as follows. The point to be noted at thisstage is the removal of the myo-inositol and neo-inositol which isdifferent from the case of the crystalline scyllo-inositol.

The soluble scyllo-inositol is dissolved together with the myo-inositol(raw material) and neo-inositol, and can be obtained as a solution bymeans of an operation such as filtration or centrifugal separation.Since the solution contains soluble peptides and salts in addition tothe myo-inositol and neo-inositol, it is purified by passing through adesalting column or activated carbon column followed by beingconcentrated to a degree at which no myo-inositol precipitates (theconcentration of the myo-inositol should be smaller than 21%). Then,crystalline scyllo-inositol to be precipitated can be isolated by anoperation such as filtration or centrifugal separation. If necessary, awater-miscible organic solvent can be added for crystallization.Examples of such an organic solvent include methanol, ethanol, andpropanol.

Meanwhile, as described hereinbelow, the scyllo-inositol may beseparated and purified by a method comprising: adding boric acid andNaCl to the obtained solution containing scyllo-inositol, to therebyform a scyllo-inositol/boric acid complex; filtrating and separating thecomplex; allowing the boric acid to be released using an acid; andcrystallizing the scyllo-inositol by adding an organic solvent such asmethanol.

4. Method of Producing Scyllo-inositol from a Liquid Mixture ContainingScyllo-inositol and Neutral Sugars Other than Scyllo-inositol

Furthermore, the present invention relates to a method of producing thescyllo-inositol from a liquid mixture containing the scyllo-inositol andneutral sugars other than the scyllo-inositol.

<4-1>

An embodiment of the method of the present invention is a method ofproducing a purified scyllo-inositol, comprising: a first step offorming a scyllo-inositol/boric acid complex by adding boric acid and ametal salt in an amount twice or more than that of the scyllo-inositoldissolved in a liquid mixture containing the scyllo-inositol and neutralacid other than the scyllo-inositol and by adjusting the pH of theliquid mixture to 8.0 to 11.0; a second step of separating the complexfrom the liquid mixture; a third step of cleaving into scyllo-inositoland boric acid by dissolving the separated complex in an acid; and afourth step of isolating and purifying the scyllo-inositol from anacidic solution or an acidic suspension obtained from the third step.

The first step of the production method is a step of forming ascyllo-inositol/boric acid complex by adding boric acid and a metal saltin an amount twice or more than that of the scyllo-inositol dissolved ina liquid mixture containing the scyllo-inositol and neutral acid otherthan the scyllo-inositol; and adjusting the pH of the liquid mixture to8.0 to 11.0.

As used herein, the “liquid mixture containing scyllo-inositol andneutral sugars other than the scyllo-inositol” may be a solution or asuspension. Further, it may be one which further contains substancesother than the “scyllo-inositol and neutral sugars other than thescyllo-inositol”, or may be one which already contains a small amount ofthe scyllo-inositol/boric acid complex. Preferably, the neutral sugarsto be contained in the liquid mixture include neutral sugars such astetrose, pentose, hexose, and heptose. Examples thereof include: aldosesuch as glucose, fructose, and galactose; ketose; various isomers ofinositol; and polyalcohols such as glycerol and ethylene glycol. Here,examples of the isomers of inositol include: myo-inositol,D-chiro-inositol, L-chiro-inositol, epi-inositol, muco-inositol,allo-inositol, cis-inositol, and neo-inositol.

Of those, myo-inositol can be particularly preferably used. In thiscase, the “liquid mixture containing scyllo-inositol and neutral sugarsother than the scyllo-inositol” includes, for example, a liquid mixturecontaining scyllo-inositol and myo-inositol which is obtained byreduction of scyllo-inosose as described hereinbelow.

For the scyllo-inosose to be used for the reduction reaction, forexample, one obtained by oxidizing myo-inositol using a microorganism ina medium or solution can be used (JP-A-2003-102492). The scyllo-inososeobtained by the microbial oxidation may be used by dissolving thepurified one, or a cultured filtrate thereof may be used. Meanwhile,scyllo-inosose prepared by oxidizing myo-inositol using a platinumcatalyst can be used.

A reducing agent to be used for reduction of the scyllo-inosose into thescyllo-inositol is not particularly limited as long as it is a reducingagent capable of reducing scyllo-inosose into scyllo-inositol in water.Examples thereof include sodium borohydride, lithium borohydride,potassium borohydride, trimethoxy sodium borohydride, and cyanatedsodium borohydride.

The reduction reaction of the scyllo-inosose can be performed, forexample, by adding a powder or solution of a reducing agent to asolution containing the scyllo-inosose dissolved at a concentration of20% or less (w/v). The solution is preferably stirred at this time. Theheat of reaction may generate owing to the reduction reaction, thereforethe reaction solution is preferably controlled to have a temperature of50° C. or lower in order to prevent decomposition of the generatedinosose. Furthermore, when a reducing agent such as sodium borohydrideis used, a part of the reducing agent may be decompose to generate ahydrogen gas. Therefore, an antifoaming agent or the like is preferablyadded to reduce foam formation of the hydrogen gas.

In a liquid mixture of the scyllo-inositol and myo-inositol obtainedfrom the reduction of the scyllo-inosose, the scyllo-inositol starts togradually crystallize when its concentration exceeds about 1.6% (w/v).Generally, reduction of 5% (w/v) scyllo-inosose solution generates about3% (w/v) of the myo-inositol and about 2% (w/v) of the scyllo-inositol.However, when the solution is left at room temperature for severalhours, about 0.4% of an oversaturated part of the scyllo-inositol startsto crystallize. Therefore, when the liquid mixture of thescyllo-inositol and myo-inositol obtained from the reduction of thescyllo-inosose is used, the step of forming a scyllo-inositol/boric acidcomplex is preferably performed prior to the generation of the crystalof the scyllo-inositol itself. The step of forming ascyllo-inositol/boric acid complex is preferably performed immediatelyafter the reduction reaction of the scyllo-inosose.

The first step is performed by adding boric acid and metal salts into a“liquid mixture containing scyllo-inositol and neutral sugars other thanthe scyllo-inositol” such as the above-described “liquid mixturecontaining scyllo-inositol and myo-inositol” in an amount twice or moremoles, preferably twice or more moles but three times or less moleslarger than that of the scyllo-inositol dissolved in the liquid mixture,respectively, and after they are dissolved, adjusting the liquid mixtureto be alkaline at pH of 8.0 to 11.0, preferably pH of 9.0 to 10.0. Theterm “twice moles” as used herein refers to a number of moles of twice.The pH of the reaction solution can be adjusted using a base such asNaOH, KOH, Na₂CO₃, or K₂CO₃.

Here, examples of metal salts to be added include one or more kindsmetal salts selected from the group consisting of NaCl, NaHCO₃, Na₂CO₃,Na₂SO₄, NaHSO₄, NaH₂PO₄, Na₂HPO₄, Na₃PO₄, borax, KCl, KHCO₃, K₂CO₃,K₂SO₄, KHSO₄, KH₂PO₄, K₂HPO₄, K₃PO₄, MgCl₂, MgCO₃, and MgSO₄. The amountof boric acid to be added, or total amount of boric acid if the liquidmixture already contains boric acid, is twice or more moles, preferablytwice or more but three times or less moles that of the dissolvedscyllo-inositol.

The first step is preferably carried out with stirring for efficientlydissolving the boric acid and metal salts in the liquid mixture andmaking the solution homogenous upon the adjustment of pH. The step ispreferably performed at a temperature ranging from 5° C. to 85° C.,preferably 15 to 40° C. Time needed for the step is not particularlylimited as long as a required amount of the scyllo-inositol/boric acidcomplex is formed, however, 12 to 76 hours are preferable in order tocollect it at an yield of 90% or higher.

Most of the scyllo-inositol/boric acid complex exists as a precipitatein the liquid mixture since it has solubility of 0.01% (w/v) or less towater as confirmed by means of NMR. In the second step, thescyllo-inositol/boric acid complex is separated from the liquid mixture.A general solid separation operation can be applied to the step, forexample, a filtration operation or centrifugal separation operation maybe applied. The scyllo-inositol left in the liquid mixture in which thescyllo-inositol/boric acid complex has been separated by the above stephas a concentration of 0.2% (w/v) or less. Therefore, most of thescyllo-inositol in the liquid mixture before the initiation of thereaction can be collected in a form of the scyllo-inositol/boric acidcomplex. Meanwhile, neutral sugars such as the myo-inositol exist in adissolved state in a solution. Therefore, the neutral sugars exist in afiltrate upon a filtration operation, and thus the neutral sugars andthe scyllo-inositol can be separated by the step.

The separated scyllo-inositol/boric acid complex can be dried andisolated as a powder. If necessary, it can be also isolated as a crystalby means of recrystallization using hot water.

Next, the third step involves dissolution of the separatedscyllo-inositol/boric acid complex into an acid. The dissolution cleavesthe scyllo-inositol/boric acid complex into scyllo-inositol and boricacid, and metal ions bound to the complex also dissociate therefrom inthe solution. The kind of an acid to be used for the dissolution in thestep is not particularly limited as long as it can dissolve the complex,however, an acid which does not form a salt having a low solubilitydepending on the kind of the metal ion is desirable. Preferably, mineralacids such as hydrochloric acid, sulfuric acid, nitric acid, andphosphoric acid can be used, and hydrochloric acid can be morepreferably used. Since those acids each gives rise to a neutralizationreaction with the metal ions generated by the dissolution, it ispreferably adjusted so that a solution containing thescyllo-inositol/boric acid complex finally becomes an acidic solution of0.1 N or higher. Also, to efficiently dissolve the scyllo-inositol/boricacid complex, the complex is preferably dissolved with an acid of 1 N orhigher to finally make an acidic solution of 0.1 N or higher.

The fourth step involves isolation and purification of thescyllo-inositol from the acidic solution or acidic suspension obtainedfrom the third step. The method of isolating and purifying thescyllo-inositol from the acidic solution is not particularly limited.However, for example, a method comprising using a resin such as an ionexchange resin as described hereinbelow or a method utilizing thedifference in solubility to an organic solvent can be used.

Further, a method comprising vacuum distillation as an ester of a loweralcohol and boric acid by adding a lower alcohol after releasing boricacid may be used (Journal of Organic Chemistry, vol. 23, p. 329-330,1958).

Of those methods, the method of isolating and purifying thescyllo-inositol using an ion exchange resin can be performed as follows.In this case, the solution obtained from the third step is preferably anacidic solution of 0.1 N or higher in which the complex completelydissolves therein. Also, the acidic solution is preferably prepared byadding an acid in an amount such that a ratio of thescyllo-inositol/boric acid complex therein becomes 2.5 (w/v) % or less,in order not to precipitate free scyllo-inositol.

First, such acidic solution is brought into contact with a strong acidicion exchange resin to thereby remove metal ions. The strong acidic ionexchange resin to be used is not particularly limited as long as itadsorbs the metal ions, and an example thereof includes an ion exchangeresin having a sulfate group. An example thereof includes Duolite C20 H⁺type (manufactured by Sumitomo Chemical Co., Ltd.). The contact may beperformed by an operation comprising batch-wise addition of the strongacidic ion exchange resin into a given amount of the solution and thenstirring. However, it is preferable that the solution is passed throughthe strong acidic ion exchange resin loaded in a column.

Next, the solution from which the metal ions are removed by means of thestrong acidic ion exchange resin is brought into contact with a strongbasic ion exchange resin or a boric acid-adsorbing resin in order toremove boric acid. Those resins are not particularly limited as long asthey adsorb boric acid. An example of the strong basic ion exchangeresin includes a resin having a quaternary ammonium group, and anexample of the boric acid-adsorbing resin includes a resin having anN-methylglucamine group. A specific example of the strong basic ionexchange resin includes Duolite A116 OH⁻ type (manufactured by SumitomoChemical Co., Ltd.). A specific example of the boric acid-adsorbingresin includes Duolite ES371N (manufactured by Sumitomo Chemical Co.,Ltd.). The contact may be performed by an operation comprisingbatch-wise addition of the ion exchange resin into a given amount of thesolution and then stirring. However, it is preferable that the solutionis added to the ion exchange resin loaded in a column.

The order of the resins with which the solution is contacted is notrandom because the boric acid and scyllo-inositol dissociate from eachother in an acidic state. The solutions are contacted with the strongacidic ion exchange resin, the strong basic ion exchange resin, and theboric acid-adsorbing resin, in this order.

A solution in which the metal ions and boric acid are removed by beingcontacted with those resins contains only scyllo-inositol that is aneutral sugar. Therefore, by concentrating the solution using a generalmethod to precipitate the scyllo-inositol, a crystal or powder of thepurified scyllo-inositol can be isolated.

Further, in the fourth step, in a case where the scyllo-inositol isisolated and purified using the difference in solubility to an organicsolvent, it can be performed as follows. In the method, a solutionobtained by the dissolution with the acid used in the third step may bea dissolved solution or a suspension because the purification operationusing the ion exchange resins or the like is not performed until theaddition of the organic solvent after the dissolution. In the thirdstep, in order to facilitate the scyllo-inositol to be precipitatedafter dissolution, the acid is preferably added in such an amount thatthe ratio of the scyllo-inositol/boric acid complex is 2.5% (w/v) ormore, preferably 3.0% to 10% (w/v), more preferably 4.0% to 6.0% (w/v).

First, a water-soluble organic solvent is added to the obtained acidicsolution or suspension to allow free scyllo-inositol to be precipitated.The organic solvent to be used is not particularly limited as long as itis a solvent which allows the scyllo-inositol to be precipitated whileboric acid is dissolved and metal salts consisting of the acid and thesalt are dissolved. Examples of such an organic solvent include ethanoland methanol.

The amount of the organic solvent is as follows: when ethanol is used,ethanol is preferably added in an amount 0.3 to 3 times larger, morepreferably 0.6 to 1.5 times larger than that of the acidic solution.When methanol is used, methanol is preferably added in an amount 0.3 to5 times larger, more preferably 0.9 to 2 times larger than that of theacidic solution. In particular, the organic solvent is efficiently addedin the above amount when the metal salt to be used to form thescyllo-inositol/boric acid complex is one or more of NaCl, NaHCO₃, andNa₂CO₃. In addition, a liquid mixture added with the aqueous organicsolvent is preferably adjusted to be an acidic solution of 0.1 N ormore.

In the fourth step, when a mixture is a homogenous solution, it is notnecessary to perform stirring, but when a mixture is a suspension, it ispreferable to perform stirring. The temperature of mixing is notparticularly limited as long as it is a temperature at which onlyscyllo-inositol precipitates, however a temperature of −10° C. to 50° C.is preferable and a temperature of 4° C. to 35° C. is more preferable.The time of mixing is preferably 10 minutes to 24 hours, more preferably3 to 5 hours.

Such operation allows only scyllo-inositol to be precipitated. Theprecipitated scyllo-inositol can be separated from the solution by meansof filtration or a general solid-liquid separation operation such ascentrifugal separation. Thus obtained scyllo-inositol is pure, however,the scyllo-inositol can be obtained as a crystal by means of a methodsuch as recrystallization if necessary. The precipitated scyllo-inositolmay be further purified using an ion exchange resin or the like afterbeing dissolved in water, to thereby increase its purity.

<4-2> Method of Producing Scyllo-inositol from Scyllo-inosose withoutGoing Through Scyllo-inositol/boric Acid Complex

Next, another embodiment will be described which comprises a method ofproducing scyllo-inositol from a liquid mixture containing thescyllo-inositol and neutral sugars other than the scyllo-inositol.

This method is a method of producing scyllo-inositol comprising: a firststep of obtaining a liquid mixture containing myo-inositol andscyllo-inositol by reducing scyllo-inosose using a metal salt of boronhydride in a solution containing scyllo-inosose; a second step ofdissolving a scyllo-inositol/boric acid complex in the liquid mixture byadding an acid to the liquid mixture and of adjusting the solution to bean acidic solution of 0.01 N or more; and a third step of allowing onlyscyllo-inositol to be precipitated by adding a water-soluble organicsolvent to the acidic solution in such an amount that myo-inositol isnot precipitated.

When scyllo-inosose is reduced using a metal salt of boron hydride in asolution containing the scyllo-inosose, boric acid and a metal ion existas well as scyllo-inositol and myo-inositol which are generated by thereduction. Therefore, a part of the scyllo-inositol forms awater-insoluble scyllo-inositol/boric acid complex, to thereby reduceits yield when the scyllo-inositol is purified only from the solutioncomponent. An object of a second embodiment of the present invention isto precipitate and purify only scyllo-inositol from an acidic solutionobtained by dissolving into an acid the scyllo-inositol/boric acidcomplex which has been generated in a small amount in a liquid mixturecontaining myo-inositol and scyllo-inositol obtained by the reduction ofthe scyllo-inosose.

In a first step, the “solution containing scyllo-inosose” includes, forexample, a solution obtained by oxidizing myo-inositol using amicroorganism in a medium or solution (JP-A-2003-102492). Scyllo-inososeobtained by the microbial oxidation may be used in a dissolved stateafter being purified, or a culture filtrate may also be used. The“solution containing scyllo-inosose” may further contain substancesother than the scyllo-inosose, such as a culture filtrate. In addition,there may be used a scyllo-inosose obtained by dissolving ascyllo-inosose which has been prepared by oxidizing myo-inositol with aplatinum catalyst.

The metal boron hydride to be used for the reduction is not particularlylimited as long as it is a reducing agent capable of reducingscyllo-inosose into scyllo-inositol in an aqueous system and capable ofreleasing boron. Examples thereof include sodium borohydride, lithiumborohydride, and potassium borohydride.

The reduction reaction of scyllo-inosose can be performed, for example,by adding a powder or solution of a reducing agent to a solutioncontaining the scyllo-inosose dissolved at a concentration of 20% orless (w/v). The solution is preferably stirred at this time. The heatmay generate owing to the reduction reaction, therefore the reactionsolution is desirably controlled to have a temperature of 50° C. orlower in order to prevent decomposition of the generated inosose.Furthermore, a part of the reducing agent may be decomposed to generatea hydrogen gas. Therefore, an antifoaming agent or the like ispreferably added to reduce foam formation of the hydrogen gas.

As described above, scyllo-inosose is reduced into scyllo-inositol andmyo-inositol and the scyllo-inositol and myo-inositol exist in asolution in a mixed state. In this case, the scyllo-inositol starts togradually crystallize when its concentration exceeds about 1.6% (w/v).Typically, reduction of 5% (w/v) of scyllo-inosose solution generatesabout 3% of myo-inositol and about 2% (w/v) of scyllo-inositol. Inaddition, when the solution is left at room temperature for severalhours, about 0.4% (w/v) of an oversaturated portion of thescyllo-inosose starts to be crystallized. Furthermore, a liquid mixturecomposed of scyllo-inositol and myo-inositol which is obtained by thereduction of the scyllo-inosose also contains boric acid, therefore apart of the scyllo-inositol starts to from a scyllo-inositol/boric acidcomplex. In the production method according to the second embodiment ofthe present invention, the second step may be performed immediatelyafter the first step, or the second step may be performed after beingleft for a while after the first step since the scyllo-inositol/boricacid complex is dissolved by an acid treatment.

In the second step, a “liquid mixture containing myo-inositol andscyllo-inositol” obtained in the first step is added with an acid todissolve a scyllo-inositol/boric acid complex in the liquid mixture, andthen the solution is adjusted to be an acidic solution of 0.01 N ormore. In this case, a mineral acid such as hydrochloric acid, sulfuricacid, nitric acid, or phosphoric acid can be used as the acid.Hydrochloric acid or sulfuric acid is preferably used.

In the third step, the acidic solution obtained in the second step isadded with a water-soluble organic solvent in such an amount that onlyscyllo-inositol is precipitated while myo-inositol is not precipitated.The aqueous organic solvent to be used at this time is not particularlylimited as long as it is an organic solvent which enables theprecipitation of scyllo-inositol and maintains the state wheremyo-inositol is dissolved. Ethanol, methanol, or 1-propanol ispreferable.

The amount such that only scyllo-inositol is precipitated whilemyo-inositol is not precipitated means an amount of 0.2 to 0.4 times forethanol, 0.2 to 0.8 times for methanol, or 0.2 to 0.4 times for1-propanol as compared to the amount of the acidic solution. Preferably,the amount is 0.35 to 0.45 times for ethanol, 0.45 to 0.55 times formethanol, or 0.35 to 0.45 times for 1-propanol as compared to the amountof the acidic solution.

When a water-soluble organic solvent is mixed, if a mixture is ahomogenous solution, it is not necessary to perform stirring, but if themixture is a suspension, it is preferable to perform stifling. Thetemperature upon the mixing is not particularly limited as long as it isa temperature at which only scyllo-inositol is precipitated, however atemperature of −10° C. to 50° C. is preferable and a temperature of 4°C. to 35° C. is more preferable. The time of mixing is preferably 15 to76 hours, more preferably 20 to 24 hours.

By adding a water-soluble organic solvent under such condition, onlyscyllo-inositol is precipitated. Thus precipitated scyllo-inositol canbe extracted as a solid by means of filtration or a common solid-liquidseparation operation such as centrifugal separation. The solid iscomposed of almost pure scyllo-inositol, however, it can be alsoobtained as a crystal by a method such as recrystallization ifnecessary. The precipitated scyllo-inositol may be further purifiedusing an ion exchange resin or the like after being dissolved in water,to further increase its purity.

EXAMPLES

Hereinafter, the present invention will be specifically described byreferring to examples.

Example 1 Method of Producing Scyllo-inositol (Small Scale)

3 L of a liquid medium containing 10.0% myo-inositol, 1.0% yeastextract, and 1.0% sucrose was adjusted to pH 7.0 with 1N NaOH, and themedium was dispensed in 100 ml aliquots into 30 pieces of 500 ml-volumebaffled conical flasks, followed by sterilization using an autoclave.One platinum loop of a slant culture of Acetobacter sp. AB10281 strain(FERN BP-10119) was inoculated to each of the conical flasks, and themicroorganism was cultured at 27° C. for 5 days in a rotary shaker.After the culture, 250 ml of water was added to each of the conicalflasks, and the mixture was stirred for 1 hour in a rotary shaker todissolve crystalline scyllo-inositol generated in the culture solution.The culture solution was collected and centrifuged (8,000 rpm 20minutes), and the obtained supernatant was defined as a culturesupernatant solution (10.2 L).

The culture supernatant solution was analyzed by high-performance liquidchromatography under the following conditions. The result revealed that12.6 mg/ml (129 g, conversion rate 43%) of scyllo-inositol was generatedin the culture supernatant solution. In the culture supernatantsolution, 2.1 mg/ml of scyllo-inosose remained, while myo-inositol wasnot detected.

The analysis conditions of high-performance liquid chromatography are asfollows.

Column: Wakosil 5NH₂ (4.6×250 mm)

Column temperature: 40° C.

Detector: RI DETECTOR ERC-7515A (ERMA CR. INC.)

Injection volume: 5 μl

Solvent: Acetonitrile-Water=4:1

Flow rate: 2 ml/min

Elution time Scyllo-inosose; 11.6 minutes

-   -   Myo-inositol; 17.8 minutes    -   Scyllo-inositol; 18.2 minutes

The above-described conversion rate of scyllo-inositol was calculated bythe following equation.

Conversion rate(%)={(Number of moles of scyllo-inositol in culturesupernatant)/(Number of moles of myo-inositol before culture)}×100

Next, the culture supernatant solution was passed through a columnformed by connecting a column (inner diameter 5 cm, length 40 cm) filledwith 500 ml of a strong acidic cation exchange resin, Duolite(registered trademark) C-20 (H⁺ type) (manufactured by Sumitomo ChemicalCo., Ltd.) with a column (inner diameter 5 cm, length 16 cm) filled with200 ml of activated carbon, and then 500 ml of ion-exchanged water waspassed through the column to wash it. The flow-through solution and thewashing solution were then passed through a column (inner diameter 7 cm,length 40 cm) filled with 1,000 ml of a strong basic anion exchangeresin, Duolite (registered trademark) A116 (OH⁻ type) (manufactured bySumitomo Chemical Co., Ltd.), and then 1,000 ml of ion-exchanged waterwas passed through the column to wash it. It was found that the obtainedflow-through solution and the water-washing solution included fewimpurities other than the above-described scyllo-inositol.

The solution obtained above was concentrated to about 700 ml underreduced pressure, and 3-fold volume of ethanol was added thereto. Then,the mixture was allowed to stand at 5° C. overnight, and the resultantcolorless crystals of pure scyllo-inositol were filtered and dried, tothereby yield 118 g of scyllo-inositol. The purification recovery yieldwas 92%, and the total recovery rate of scyllo-inositol frommyo-inositol was 39%.

Example 2 Method of Producing Scyllo-inositol (Large Scale)

40 L of a liquid medium containing 10.0% myo-inositol, 1.0% yeastextract, and 1.0% sucrose was poured in a 50-L jar fermentor andadjusted to pH 7.0 with 1N NaOH, followed by sterilization using anautoclave. 400 ml of Acetobacter sp. AB10281 (FERM BP-10119), which hadbeen cultured in a medium having the same composition (conical flask),was inoculated and cultured at 27° C. for 5 days at an aeration rate of1 vvm and an agitation of 200 rpm. After the culture, 60 L of hot water(about 50° C.) was added to about 40 L of the collected culturesolution, and the mixture was stirred for 1 hour to dissolve crystallinescyllo-inositol which had been present in the culture solution. Theculture solution was subjected to continuous centrifugation (8,000 rpm)to remove the cells, and the resultant solution was defined as asolution from which the cultured microorganism had been removed (about100 L).

The solution from which the cultured microorganism had been removed wasanalyzed by high-performance liquid chromatography. The result revealedthat 16.8 mg/ml (1.68 kg, conversion rate 42%) of scyllo-inositol wasgenerated in the solution from which the cultured microorganism had beenremoved. In the solution from which the cultured microorganism had beenremoved, 2.9 mg/ml of scyllo-inosose remained, while myo-inositol wasnot detected. The analysis condition for the high-performance liquidchromatography is the same as that of Example 1.

Next, 400 g of sodium hydroxide was added to 100 L of the obtainedsolution, and the mixture was heated with stirring at 98° C. for 1 hour.Subsequently, 560 g of sodium hydroxide, 1,340 g of boric acid, and1,260 g of NaCl were added and dissolved while the mixture was hot.After the stirring was stopped, the heat of the solution was released,and the solution was allowed to stand until the temperature reached 23°C. (about 24 hours).

Next, the solution was filtered to isolate crystals of ascyllo-inositol/boric acid complex formed in the liquid, and thecrystals were washed with water until they became white. The resultantcrystals (about 3.9 kg) were taken out to another container, and 5.9 Lof water and 1.95 L of 37% hydrochloric acid were added thereto,followed by stirring. 30 minutes later, to precipitate scyllo-inositolwhich was released from boric acid by such procedure, 9.4 L of methanolwas added, and the mixture was further stirred for 1 hour.

Next, the solution was filtrated to isolate crystallized scyllo-inositolin the liquid, and the crystals were washed with 1 L of 50% methanol.The resultant fine powder crystals (about 1.8 kg) were taken out toanother container, and 10 L of water was added thereto, followed byboiling with stirring for 1 hour. Thereafter, the solution was cooled to20° C. with stirring and then filtered, to thereby yield fine crystalsof scyllo-inositol. After drying, 1.35 kg of colorless crystal of purescyllo-inositol was obtained. The purification recovery yield was 80%,while the total recovery rate of scyllo-inositol from myo-inositol was34%.

Example 3 Identification of Scyllo-inositol-producing MicroorganismsBased on the Nucleotide Sequence of 16SrRNA

The nucleotide sequences of 16SrRNA were analyzed for 4 microbialstrains consisting of 3 natural isolated strains, AB10285, AB10286, andAB10287, each having an ability to convert myo-inositol intoscyllo-inositol, and AB10281 obtained from AB10253, in accordance withthe conventional method. More specifically, a genomic DNA was extractedfrom the cultured cells, and then corresponding DNA fragments wereprepared by the PCR using primers that were designed so as to amplifyabout 1.6 kbp of 16SrRNA, followed by analysis of about 1.3 kbp ofsequences (Hokkaido System Science Co., Ltd.). The results of thesequences were inquired against database to identify related species.

Table 2 shows the inquiry results, homologies, and conversion rates intoscyllo-inositol in culturing the microorganisms in the same way asExample 1.

Identification of Microbial Strains by Analysis of 16SrRNA NucleotideSequence

TABLE 2 Strain Conversion name Name of identified microorganismsHomology rate AB10281 Acetobacter cerevisiae, 99.93% 40.0% Acetobactermalorum AB10285 Acetobacter cerevisiae, 99.78% 4.5% Acetobacter malorumAB10286 Burkholderia andropogonis 98.12% 2.6% AB10287 Burkholderiaandropogonis 98.04% 1.4%

The results revealed that 4 microbial strains each having an ability toconvert myo-inositol into scyllo-inositol may be broadly divided into 2groups. As the first group, AB10281 and AB10285 were identified asAcetobacter cerevisiae or Acetobacter malorum, while the second group,AB10286 and AB10287, were identified as Burkholderia andropogonis.

Example 4 Isolation of NAD⁺-Independent Myo-inositol 2-dehydrogenasefrom Acetobacter sp. AB10253

3 g of myo-inositol, 1 g of yeast extract (FNI205: manufactured byLallemand BI), and 0.5 g of glucose were added to 500 ml-volume baffledconical flask and dissolved in water so that the mixture has a volume of100 ml, and the solution was adjusted to pH 5.0, followed bysterilization using an autoclave. According to such procedures, 4 piecesof flasks of a medium were prepared. One platinum loop of Acetobactersp. AB10253 from a slant was added to each medium, and the microorganismwas precultured at 27° C. for 2 days using a rotary shaker.

Next, 1.2 kg of myo-inositol, 0.4 kg of yeast extract (FNI205:manufactured by Lallemand BI), and 0.2 kg of glucose were added to 50-Ljar fermentor and dissolved in water so that the mixture has a volume of40 L. The solution was adjusted to pH 5.0, followed by sterilizationusing an autoclave. About 400 ml of a microbial solution of theprecultured Acetobacter sp. AB10253 was added thereto, and themicroorganism was cultured at 27° C. for 3 days at an aeration rate of 1vvm and an agitation of 200 rpm.

After the culture, the cells were obtained as precipitates using acontinuous centrifugator. The obtained cells were resuspended in 2 L ofwater, and washed cells were obtained by centrifugation and suspended in2 L of 20 mM Tris buffer (pH 7.0). Next, ultrasonic waves were appliedto the suspension to disrupt the cells. The cell lysis solution wascentrifuged to precipitate the disrupted cells, and the disrupted cellswere obtained as precipitates. The precipitates were suspended by adding500 ml of 20 mM Tris buffer (pH 7.0), 0.6% Triton X-100 (manufactured byKodak), and enzymes were extracted at 15° C. for 3 hours. Thereafter,the suspension was centrifuged, and 420 ml of the supernatant (crudeenzyme solution) was taken out.

420 ml of the crude enzyme solution was concentrated to 150 ml using anultrafilter (MW 30,000 cut off), and the concentrated solution waspassed through a 400 ml-DEAE Toyopearl column equilibrated with 20 mMTris buffer (pH 7.0) to adsorb proteins. Next, a solution with a linearconcentration gradient from 0 mM to 500 mM NaCl in 20 mM Tris buffer (pH7.0) containing no surfactant (total volume 1.6 L) was passed throughthe protein-adsorbed column at a rate of 10 ml/min to elute theproteins. The eluate was fractionated into 40 ml fractions. Next, 600 mlof 20 mM Tris buffer (pH 7.0) containing no surfactant was passedthrough the column again for washing, and then a solution with a linearconcentration gradient from 0 mM to 500 mM NaCl in 20 mM Tris buffer (pH7.0) containing 0.1% Triton X-100 (total volume 1.6 L) was passedthrough the column at a rate of 10 ml/min to elute the proteins. Theeluate was fractionated into 40 ml fractions.

The enzyme activity of each fraction was measured by a standard method:that is, the change in absorbance at 600 nm of 1 ml of a solutioncontaining 50 μl of the protein solution, 100 mM phosphate buffer (pH5.0), 5 mg of myo-inositol, and 0.4 mg of 2,4-dichloroindophenol(oxidized DCIP) was calculated into a reaction rate, and an activity tooxidize 1 μmol of myo-inositol per minute was defined as one unit.

The results revealed that the target enzyme was eluted in fractions ofsolutions containing 20 mM Tris buffer (pH 7.0) containing 0.1% TritonX-100 and 100 to 170 mM NaCl. Next, those fractions (240 ml) werecollected and concentrated to 30 ml using an ultrafilter (MW 30,000 cutoff), and 100 ml of 20 mM Tris buffer (pH 7.0) containing 0.1% TritonX-100 was added thereto. The solution was further concentrated to 30 ml,and 70 ml of 20 mM Tris buffer (pH 7.0) was added to the concentratedsolution for desalting.

Next, the thus-prepared enzyme solution was passed through 100ml-hydroxyapatite column equilibrated with 20 mM Tris buffer (pH 7.0)containing 0.1% Triton X-100 to adsorb proteins. Subsequently, asolution with a linear concentration gradient from 0 mM to 500 mMphosphate buffer (pH 7.0) in Tris buffer (pH 7.0) containing 0.1% TritonX-100 (total volume 400 ml) was passed through the protein-adsorbedcolumn at a rate of 3 ml/min to elute the proteins. The eluate wasfractionated into each of 10 ml-fractions, and the enzyme activity ofeach fraction was measured.

The results revealed that the target enzyme was eluted in fractions ofsolutions containing 20 mM Tris buffer (pH 7.0) containing 0.1% TritonX-100 and 100 to 170 mM phosphate buffer. The thus-obtained enzymesolution was found to contain almost pure NAD⁺-independent myo-inositol2-dehydrogenase. Next, the fractions (40 ml) were collected andconcentrated to 5 ml using an ultrafilter (MW 30,000 cut off), and 100ml of 20 mM Tris buffer (pH 7.0) was added. The solution was furtherconcentrated to 5 ml, followed by desalting.

The thus-prepared concentrated solution was again passed through 20ml-DEAE Toyopearl column (manufactured by Tosoh Corporation)equilibrated with 20 mM Tris buffer (pH 7.0) containing 0.1% TritonX-100 to adsorb proteins. Next, a solution with a linear concentrationgradient from 50 mM to 250 mM NaCl in 20 mM Tris buffer (pH 7.0)containing 0.1% Triton X-100 (total volume 160 ml) was passed throughthe protein-adsorbed column at a rate of 1 ml/min to elute the proteins.The eluate was fractionated into each of 4 ml-fractions. After thefractionation, the enzyme activity of each fraction was measured, andeach fraction having the activity was subjected to SDS electrophoresis.

As a result, SDS electrophoresis revealed the bands of proteins thatcorrelate with the enzyme activity of the target enzyme. Removal ofbands of proteins derived from fractions having no activity revealedthat the target enzyme was an enzyme containing at least proteins havingmolecular weights of about 76 k Dalton and about 46 k Dalton.

Meanwhile, the fractions having the enzyme activity had red color, andthe UV spectrum pattern revealed that the fractions contained cytochromeC. Moreover, the content of the target protein and the absorbance ofcytochrome C revealed that 1 mol of the target enzyme contains 1 mol ofcytochrome C.

For measurement of the optimum pH, the enzyme activity was measuredwhile changing a buffer and pH value. As the buffer, there were used 100mM phosphate buffer (pH 3 to 8), 100 mM Tris buffer (pH 7 to 8), and 100mM carbonate buffer (pH 8 to 11). The results revealed that the targetenzyme has the maximum activity at pH 4.5 to 5.5. Moreover, in measuringstandard enzyme activity (100 mM phosphate buffer (pH 5.0)), variousheavy metal ions (Sn²⁺, Mn²⁺, Mg²⁺, Cu²⁺, Fe, Zn²⁺, Co²⁺, Pb²⁺, Ca²⁺,Cd²⁺, and Ni²⁺) were added, and it was revealed that the target enzymeis specifically inhibited by Sn²⁺ ion. The enzyme activity was inhibitedin the presence of 1 mM Sn²⁺ ion to 1% or less of the activity in theabsence of Sn²⁺ ion.

Meanwhile, it was confirmed that the target enzyme is an enzymeextracted with Triton X-100 from a membrane fraction, and the extractedenzyme oxidizes myo-inositol in the presence of reduced DCIP, while nooxygen absorption occurs in the absence of reduced DCIP. The facts meanthat the enzyme is coupled to the electron transport system of the cellmembrane in a living body to deprive electrons from myo-inositol togenerate scyllo-inositol.

The substrate specificity of the target enzyme was determined bymeasuring the enzyme activity in a solution containing various sugarsinstead of myo-inositol at a final concentration of 50 mM. Meanwhile,the Km value was measured by measuring the enzyme activity for eachsugar to which this enzyme shows the activity while changing theconcentration of the sugar. Moreover, the oxidization reaction productswere analyzed by HPLC to determine what substances were generated. Themeasurement was performed under the following HPLC conditions: therewere used Wakosil 5NH₂ column φ 4.6×250 mm (column temperature 40° C.)as a column, 80% acetonitrile as a mobile phase (flow rate 2 ml/min),and RI detector as a detector.

As a result, the target enzyme was found to react with D-chiro-inositol(relative activity 100%: Km=8.8 mM), muco-inositol (relative activity68%: Km=14.5 mM), and myo-inositol (relative activity 53%: Km=20 mM) toconvert them into D-chiro-1-inosose, L-chiro-2-inosose, andscyllo-inosose, respectively. The enzyme was found not to react withallo-inositol, scyllo-inositol, L-chiro-inositol, and glucose.

Example 5 Conversion of Myo-inositol into Scyllo-inosose byNAD⁺-Independent Myo-inositol 2-dehydrogenase

In the same way as Example 4, purification was performed using a 40L-jar fermentor, and 5 ml of an enzyme solution obtained by purificationand desalting with a 100-ml hydroxyapatite column was defined as anenzyme solution, which was used in the following conversion reaction.

30 g of myo-inositol (166.7 mmol) and 15 ml of 1 M phosphate buffer (pH5.0) were added to 400 ml-centrifuge tube, and the mixture was dilutedto 300 ml with water to dissolve myo-inositol. 1 ml of an enzymesolution at 30° C., and 8 g of reduced DCIP (Na salt) was graduallyadded to the solution with stirring. After disappearance of the bluecolor derived from reduced DCIP, white insoluble matters generated withthe disappearance of the blue color (oxidized DCIP) were precipitated bycentrifugation, and the supernatant was transferred to a new 400ml-centrifuge tube. Then, the solution was adjusted to pH 5.0 with 1Nphosphoric acid, and 8 g of reduced DCIP (Na salt) was further addedwith stirring. The procedure was repeated 6 times to add a total of 48 gof reduced DCIP (Na salt), and at the time of the disappearance of theblue color, 3 g of reduced DCIP (Na salt) was finally added. The mixturewas allowed to stand for 1 hour and centrifuged, and the supernatant wastaken. Such procedures yielded 292 ml of the supernatant. The procedurestook 8 hours.

Next, the resultant supernatant was passed through a column filled with100 ml of a strong acidic ion exchange resin (Duolite C20, H⁺ type) at aflow rate of 1.5 ml/min, and the resultant eluate was passed through acolumn filled with 150 ml of a weak base ion exchange resin (Duolite368S, OH— type). Moreover, the resultant eluate was passed through acolumn filled with 50 ml of activated carbon. The resultant eluate wasconcentrated, to thereby yield 26.5 g (148.9 mmol) of white powder(yield 89%). The substance was analyzed by NM and HPLC, and thesubstance was found to contain 99% scyllo-inositol and 1% myo-inositol.

Example 6 Screening of Acetobacter sp. AB10253 from Mutants Based onNAD⁺-Independent Myo-inositol 2-dehydrogenase Activity

5 ml of a liquid medium (pH 5.0) containing 1% yeast extract(manufactured by Difco Laboratories) and 0.5% glucose in a test tube wassterilized, and one platinum loop of Acetobacter sp. AB10253 from aslant was added thereto, followed by shaking culture at 27° C. for 16hours. 2.5 ml of a culture solution was taken to a sterilized tube andcentrifuged at 3,000×g to collect the cells. The supernatant wasdiscarded, and the cells were resuspended in 2.5 ml of 200 mM phosphatebuffer solution (pH 8.0), followed by centrifugation at 3,000×g tocollect the cells. The supernatant was discarded, and the cells wereresuspended in 2.5 ml of 200 mM phosphate buffer (pH 8.0). 2.0 ml of thesuspension was poured into a sterilized 100 ml-conical flask, and 0.5 mlof 40% glucose solution and 7.5 ml of 200 mM phosphate buffer (pH 8.0)were added and mixed. To the mixture, 20 μl of ethyl methanesulfonatewas added, and shaking culture was performed at 30° C. for 45 minutes.After the treatment, 1 ml of the mixture was taken out to a sterilizedtube and centrifuged at 3,000×g to collect cells. The supernatant wasdiscarded, and the cells were suspended in 2.5 ml of 200 mM phosphatebuffer (pH 7.0), followed by centrifugation at 3,000×g to collect cells.The supernatant was discarded, and the cells were resuspended in 2.5 mlof 200 mM phosphate buffer (pH 7.0), to thereby yield a solutioncontaining a mutation-treated microorganism.

Next, the mutation-treated Acetobacter sp. AB10253 was inoculated byspreading 0.12 ml of the solution containing the mutation-treatedmicroorganism on an agar medium prepared by solidifying a sterilizedmedium (pH 5.0) containing 3% myo-inositol, 1% yeast extract(manufactured by Difco Laboratories), 0.5% glucose, and 1.5% agar in9-cm dish, and culture was performed at 27° C. for 2 days. Thoseprocedures reduced the viable count to about 1.6%. Meanwhile, about 95to 125 colonies were formed per dish.

After the culture, over the colonies formed in the 9-cm dish, 10 ml of aviscous solution was slowly poured, which had been prepared bysterilizing with filtration a solution having a composition of 100 mMphosphate buffer, 1% myo-inositol, and 0.4% oxidized DCIP and addingthereto equal volume of 1% agar solution sterilized with an autoclavewhile it was hot, followed by cooling to 36° C. so as to inhibitsolidification of the agar. The agar medium that had been subjected tosuch treatments was slowly cooled at 27° C. to be solidified and layeredover the colonies formed in the 9-cm dish.

After the treatment, the dish was incubated at 27° C., and then it wasobserved that the blue color of oxidized DCIP that had spread all overthe agar medium gradually began to become transparent only around thecolonies depending on the degree of the NAD⁺-independent myo-inositol2-dehydrogenase activity. At that time, the colonies at the positionwhere the color more rapidly became transparent were scratched by asterilized needle and subcultured in a fresh medium. All of the 2,154colonies were subjected to a primary selection, to thereby obtain 22strains having high NAD⁺-independent myo-inositol 2-dehydrogenaseactivity.

Next, for the secondary selection, the thus-obtained 22 strains ofbacteria that formed colonies were individually inoculated to 5 ml of asterilized liquid medium (pH 5.0) containing 3% myo-inositol, 1% yeastextract (manufactured by Difco Laboratories), and 0.5% glucose in a testtube. Shaking culture was performed at 27° C. for 3 days, and then 1 mlof the culture solution was taken out to a test tube, followed bycentrifugation at 3,000×g to collect the cells. The supernatant wasdiscarded, and 1 ml of a solution containing 10% myo-inositol, 50 mMphosphate buffer (pH 5.0) was added to the test tube containing thecells, followed by shaking culture at 27° C. for 4 hours. Then,centrifugation was performed at 16,000×g, and the conversion rate ofmyo-inositol into scyllo-inosose in the supernatant was measured byHPLC. On the other hand, 0.5 ml of the culture solution (5 ml) was takenout into a sterilized tube and centrifuged at 3,000×g, and thesupernatant was discarded. The cells obtained as precipitates werewashed with water, and the NAD⁺-independent myo-inositol 2-dehydrogenaseactivity was measured.

As a result, in 3 strains (strain No. E6-55, H2-68, and B7-14) among the22 mutant strains, the NAD⁺-independent myo-inositol 2-dehydrogenaseactivity increased 1.3-fold or more after the mutation, and the activityincreased 1.6-fold, 2.2-fold, and 2.8-fold, respectively. Meanwhile, theconversion rate of myo-inositol into scyllo-inosose increased 1.1-fold,1.4-fold, and 1.5-fold, respectively, and the conversion rate ofmyo-inositol into scyllo-inosose of the strains having 1.3-fold or lessNAD⁺-independent myo-inositol 2-dehydrogenase activity was equal to thatof the microorganism before mutation. The results revealed thatscreening based on the NAD⁺-independent myo-inositol 2-dehydrogenaseactivity correlates with increase in the conversion rate of myo-inositolinto scyllo-inosose.

Example 7 Production of Scyllo-inosose by Conversion of Myo-inositolinto Scyllo-inosose Using the Mutant Strain (B7-14)

10 g of myo-inositol, 1 g of yeast extract (FNI205: manufactured byLallemand BI), and 0.5 g of glucose were added to a 500 ml-volumebaffled conical flask and dissolved in water so that the mixture has avolume of 100 ml, and the solution was adjusted to pH 5.0, followed bysterilization using an autoclave. According to such procedures, 20flasks of a medium (corresponding to 2 L: myo-inositol 200 g (1.11mmol)) were prepared. One platinum loop of the mutant strain (B7-14)from a slant was added to each medium, and the bacterium was cultured at27° C. for 3 days using a rotary shaker.

After the culture, the culture solution was centrifuged, and theresultant supernatant was passed through a column filled with 500 ml ofa strong acidic ion exchange resin (Duolite C20, H⁺ type) at a flow rateof 10 ml/min. The obtained eluate was passed through a column filledwith 900 ml of a weak base ion exchange resin (Duolite 368S, OH⁻ type),and the resultant eluate was further passed through a column filled with50 ml of activated carbon. The resultant eluate was concentrated, tothereby yield 162 g (0.91 mol) of white powder (yield 82%). Thesubstance was analyzed by NMR and HPLC, and it was found that thesubstance contained 91% of scyllo-inosose, 3% of myo-inositol, and 6% ofscyllo-inositol (scyllo-inosose of purity of 91%).

Example 8 Production of Scyllo-inositol by Conversion and ChemicalReduction of Myo-inositol into Scyllo-inosose Using the Mutant Strain(B7-14)

10 g of myo-inositol, 1 g of yeast extract (FNI205: manufactured byLallemand BI), and 0.5 g of glucose were added to a 500 ml-volumebaffled conical flask and dissolved in water so that the mixture has avolume of 100 ml, and the solution was adjusted to pH 5.0, followed bysterilization using an autoclave. According to such procedures, 20flasks of a medium (corresponding to 2 L: myo-inositol 200 g (1.11mmol)) were prepared. One platinum loop of the mutant strain (B7-14)from a slant was added to each medium, and the bacterium was cultured at27° C. for 3 days using a rotary shaker.

After the culture, the culture solution was centrifuged at 8,000×g, andabout 2 L of the resultant supernatant was adjusted to pH 7.5 with 5NNaOH solution. 9.2 g of NaBH₄ was added to the solution with stirring toperform a reduction reaction. The temperature of the reaction solutionwas raised to 37° C. by the reaction heat. 30 minutes later, insolublematter gradually appeared, and 1.2 L of water was added thereto todissolve almost all of the generated insoluble matter. The solution wasfiltered to remove the insoluble matter, and the filtrate was passedthrough a column filled with 500 ml of a strong acidic ion exchangeresin (Duolite C20, H⁺ type) at a flow rate of 10 ml/min. The resultanteluate was passed through a column filled with 900 ml of a strong baseion exchange resin (Duolite A116, OH⁻ type), and the resultant eluatewas further passed through a column filled with 300 ml of activatedcarbon. The resultant eluate was concentrated, to thereby yield 145 g ofwhite powder. The substance was analyzed by HPLC, and it was found thatthe substance contained 36% of scyllo-inositol and 64% of myo-inositol.

The resultant white powder was suspended in water so as to have a volumeof 470 ml, and the suspension was heated to 70° C. to thoroughlydissolve myo-inositol. The suspension was cooled to 30° C. withstirring, and the white solution was filtered to collect the insolublematter. The insoluble matter was washed with a small amount of water anddried, to thereby yield 44.2 g of powder. The substance was analyzed byHPLC, and it was found that the substance contained 98% ofscyllo-inositol and 2% of myo-inositol. Furthermore, 700 ml of water wasadded to the resultant powder, and the mixture was heated to 85° C. todissolve all of them. The mixture was gradually cooled to 30° C. withstirring, and 700 ml of ethanol was added thereto. The mixture wasallowed to stand overnight at room temperature, and then the resultantcrystals were collected by filtration and dried, to thereby yield 40.1 g(222.8 mmol) of crystals (yield 20%). The crystals were analyzed by NMRand HPLC, and it was found that the obtained substance wasscyllo-inositol having a purity of 99.9% or more.

Example 9 Purification of Scyllo-inositol Dehydrogenase Produced byAcetobacter sp. AB10281 FERM BP-10119

3 L of a liquid medium containing 10.0% of myo-inositol, 1.0% of yeastextract, and 1.0% of sucrose was adjusted to pH 7.0 with 1N NaOH, andthe medium was dispensed in 100 ml aliquots into 30 pieces of 500ml-volume baffled conical flasks, followed by sterilization using anautoclave. One platinum loop of a slant culture of Acetobacter sp.AB10281 FERM BP-10119 was inoculated to each conical flask, and themicroorganism was cultured at 27° C. for 5 days using a rotary shaker(180 rpm). After the culture, 250 ml of water was added to each conicalflask, and the mixture was stirred for 1 hour using a rotary shaker todissolve crystalline scyllo-inositol that had been present in theculture solution. The culture solution was collected and centrifuged(8,000 rpm, 20 minutes), to thereby yield cells (wet weight 75 g).

The cells were suspended in 300 ml of water and were disrupted byultrasonic wave at 10° C. or less. The lysis solution indicated pH 4.8,and the solution was adjusted to pH 7.0 with 1N NaOH. Then, the solutionwas centrifuged (16,000 rpm, 20 minutes) to separate the supernatant.Next, MgSO₄ was added so that the supernatant contains 2 mM Mg²⁺, andthe solution was charged onto a Blue-Toyopearl column (manufactured byTosoh Corporation: 20 ml). Then, 50 ml of 20 mM Tris buffer (pH 7.0)supplemented with 2 mM Mg²⁺ was passed through the column to wash it.Thereafter, 50 ml of 20 mM Tris buffer (pH 7.0) supplemented with 1 MKCl was passed through the column to elute adsorbed proteins. Next, theeluate was concentrated using an ultrafilter (MW 30,000 cut off), and 50ml of 20 mM Tris buffer (pH 7.0) was added to the concentrated solution,followed by concentration again, to thereby yield a desaltedconcentrated solution. Subsequently, the desalted concentrated solutionwas charged onto a DEAE Toyopearl column (Tosoh Corporation: 20 ml), andelution was performed with a solution with a linear concentrationgradient from 0 mM to 500 mM NaCl in 20 mM Tris buffer (pH 7.0). Then,the eluate was fractionated into fractions. The scyllo-inositoldehydrogenase activity of each of the fractionated solutions wasmeasured, and three fractions, an unadsorbed fraction (SIDH1), afraction eluted with 200 mM NaCl (SIDH2), and a fraction eluted with 300mM (SIDH3), were found to have the activity.

The activity was measured as follows: 5 μl of a reaction solution (200mM Tris buffer (pH 8.0), 2% of NADPH, and 1% of scyllo-inosose) and 5 μlof an enzyme solution were mixed and allowed to react at 36° C. for 30minutes, and then 500 μl of water was immediately added, followed bymeasurement of the absorbance at 340 nm. The decrease in the absorbanceat 340 nm was measured based on a blank value for a test solutionobtained by using water instead of the enzyme solution. The enzymesolution was diluted as required.

The above-described column-unadsorbed fraction was further charged ontoa CM Toyopearl column (Tosoh Corporation: 20 ml), and elution wasperformed with a solution with a linear concentration gradient from 0 mMto 500 mM NaCl in 20 mM Tris buffer (pH 7.0). Then, the eluate wasfractionated into fractions. The scyllo-inositol dehydrogenase activityin each of the fractionated solutions was measured, and the unadsorbedfraction was found to have the activity. The fraction eluted with 200 mMNaCl and the fraction eluted with 300 mM were separately desalted withan ultrafilter again, and elution was performed with a solution with alinear concentration gradient from 200 mM to 300 mM NaCl in 20 mM Trisbuffer (pH 7.0). Then, the eluate was fractionated into fractions andpurified. Then, those three enzyme solutions having the scyllo-inositoldehydrogenase activity were separately concentrated using anultrafilter, and the concentrated solutions were charged onto a gelfiltration column (Tosoh Corporation: 2000 SWXL), respectively. Then,the eluates were purified with 20 mM phosphate buffer (pH 7.0)supplemented with 200 mM NaCl. The thus-purified enzyme solutions weresubjected to slab-gel SDS electrophoresis, and the gel after theelectrophoresis was stained with a Coomassie brilliant blue stainingsolution (Rapid CBB KANTO: manufactured by Kanto Chemical Co., Inc.),and then it was decolored. The purity of the bands of interest wasmeasured by measuring the blue bands of proteins using a densitometer(manufactured by ATTO Corporation), and it was found that purity of eachfraction was 85% or more.

Example 10 Purification of the Enzyme of the Present Invention Producedby Escherichia coli K12 ATCC 10798 and Analysis of its N-Terminal

3 L of LB broth medium (1% Bacto-tryptone, 0.5% yeast extract, 1% NaCl,pH 7.0) containing 0.5% L-sorbose was dispensed in 100 ml aliquots into30 pieces of 500-ml Sakaguchi flasks, followed by sterilization using anautoclave. One platinum loop of a slant culture of Escherichia coli K12was inoculated to each conical flask, and the microorganism was culturedat 36° C. for 1 day using a recipro shaker (135 rpm). After the culture,the culture media were collected and centrifuged (8,000 rpm, 20minutes), to thereby yield cells (wet weight 32 g). The cells weresuspended in 100 ml of water and were disrupted by ultrasonic waves at10° C. or less. The lysis solution indicated pH 6.8, and the solutionwas adjusted to pH 7.0 with 1N NaOH solution and then centrifuged(16,000 rpm, 20 minutes) to separate the supernatant. Next, MgSO₄ wasadded so that the supernatant contains 2 mM Mg²⁺, and the solution wascharged onto a Blue-Toyopearl column (Tosoh Corporation: 20 ml). Then,50 ml of 20 mM Tris buffer (pH 7.0) supplemented with 2 mM Mg²⁺ waspassed through the column to wash it. Subsequently, 50 ml of 20 mM Trisbuffer (pH 7.0) supplemented with 1 M KCl was passed through the columnto elute adsorbed proteins. Next, the eluate was concentrated using anultrafilter (MW 30,000 cut off), and 50 ml of 20 mM Tris buffer (pH 7.0)was added to the concentrated solution, followed by concentration again,to thereby yield a desalted concentrated solution. Next, the desaltedconcentrated solution was charged onto a DEAE Toyopearl column (TosohCorporation: 20 ml), and elution was performed with a solution with alinear concentration gradient from 0 mM to 500 mM NaCl in 20 mM Trisbuffer (pH 7.0). The eluate was fractionated into fractions. Thescyllo-inositol dehydrogenase activity of each of the fractionatedsolutions was measured, and it was found that the fraction eluted with300 mM had the activity.

The activity measurement was performed in the same way as in the aboveExample 9, and the decrease in the absorbance at 340 nm was measured.The enzyme solution was diluted as required.

The fraction eluted with 300 mM NaCl was desalted using an ultrafilteragain, and the resultant was charged onto a DEAE Toyopearl column (TosohCorporation: 20 ml). Then, elution was performed with a solution with alinear concentration gradient from 250 mM to 350 mM NaCl in 20 mM Trisbuffer (pH 7.0), and purification was performed by repeating theoperation of fractionating the eluate three times to remove contaminantproteins. Furthermore, the enzyme solution having the scyllo-inositoldehydrogenase activity was concentrated using an ultrafilter, and theconcentrated solution was charged onto a gel filtration column (TosohCorporation: 2000 SWXL). The eluate was purified with 20 mM phosphatebuffer (pH 7.0) supplemented with 200 mM NaCl.

The thus-purified enzyme solution was subjected to slab-gel SDSelectrophoresis, and then the gel was taken out, and the proteins weretransferred to a PVDF membrane (Immobilon PSQ: manufactured by MilliporeCorporation) having the same size as that of the gel using a semidryelectroplotter (manufactured by Funakoshi Co., Ltd.). Then, the PVDFmembrane was taken out and stained with a Coomassie brilliant bluestaining solution (Rapid CBB KANTO: manufactured by Kanto Chemical Co.,Inc.), and then it was decolored. The purity was measured using adensitometer in the same way as Example 9, and the purity was 40%.Moreover, the portion corresponding to the target protein was cut off toremove unwanted proteins present around the portion, to thereby yield anenzyme of the present invention having a high purity.

Next, the enzyme of the present invention having a high purity whichexists on the PVDF membrane was analyzed using an N-terminal amino acidanalyzer (Hewlett-Packard Company). As a result, a sequence ofserine-aspartic acid-asparagine-isoleucine-arginine was detected. DNAencoding a protein having such a sequence was searched from the databaseon the entire sequence of Escherichia coli (database name “Colibri”),and the ydgJ gene (or b1624 gene) was matched. The gene product of theydgj gene had been predicted as one of oxidoreductases, but itssubstrate and product were unknown.

Example 11 Isolation and Expression of the DNA of the Present InventionDerived from Escherichia coli K12 ATCC10798>

To obtain the ydgJ gene that was assumed to encode the enzyme of thepresent invention, first, the entire genome of Escherichia coli K12 tobe used as templates was extracted as follows. One platinum loop ofEscherichia coli K12, which had been cultured in LB slant medium (1%bacto-tryptone, 0.5% yeast extract, 1% NaCl, pH 7.0, 1.5% agar), wasinoculated to 100 ml of LB flask medium (1% bacto-tryptone, 0.5% yeastextract, 1% NaCl, pH 7.0) and aerobically cultured for 8 hours at 36°C., followed by collection of cells. To the pellet of the cells wereadded 15 ml of Saline-EDTA solution (0.15 M NaCl, 0.1 M EDTA, pH 8.0)and 50 mg of lysozyme, and the cells were suspended, followed byreaction at 37° C. for 2 hours. After the treatment, 0.5 ml of 25% SDSsolution was added to the solution to completely lyse the cells, and 3ml of phenol was added to denatured proteins, followed bycentrifugation. The supernatant was taken out, and 20 ml of 2-propanolwas added to the solution to yield crude genomic DNA. The yielded crudegenomic DNA was precipitated by centrifugation, and the supernatant wasremoved, followed by drying under reduced pressure. The dried crudegenomic DNA was further dissolved in 3 ml of TE solution (10 mMTris-HCl, 1 mM EDTA, pH 8.0), and then 0.01 mg of RNase was added,followed by reaction at 36° C. for 2 hours to degrade RNA. Then, 0.01 mgof proteinase K was added, and the mixture was allowed to react at 36°C. for 2 hours to degrade proteins. Next, 1 ml of phenol-chloroformmixed solution (1:1) was added, and the mixture was slowly stirred todenature RNAase and proteinase K. The mixture was separated into twophases by centrifugation, and the upper layer (an aqueous phase) wastaken out and was adjusted to pH 5.2 by adding 0.3 ml of 3 M sodiumacetate solution. To the solution was added with 3 ml of 2-propanol toyield genomic DNA. The obtained genomic DNA was precipitated bycentrifugation, and the supernatant was removed, followed by dryingunder reduced pressure. The dried genomic DNA was dissolved in 3 ml ofTE solution, and the process from the operation of adding 1 ml of thephenol-chloroform mixed solution (1:1) to the operation of dissolvingDNA in 3 ml of the TE solution was repeated again, followed bycentrifugation. In the same way as above, equal volume of 2-propanol wasadded to the supernatant at pH 5.2, to thereby prepare a solution ofgenomic DNA of Escherichia coli K12. The thus-obtained genomic DNA wasused as a template DNA solution for PCR.

A fragment that is derived from Escherichia coli K12 and contains aribosome binding site (RBS) of the ydgJ gene was cloned, and PCR wasperformed using primers having the following sequences to express thegene as a recombinant enzyme.

SEQ ID No. 15: ydgj-F 5′-cattcaagottaatgagaggcaatgacatgagcg-3′ SEQ IDNo. 16: ydgj-R 5′-tcggaattcttcatgcaaggcacaaagtcgc-3′

For PCR, Ex taq reaction solution from Takara Shuzo Co., Ltd. was used,and a solution having a composition of 5 μl of 10× Takara ExTaq Buffer,4 μl of dNTP mixture, 30 ng of the template DNA, 1 μl of 10 μM primersolution, and 0.5 μl of Takara ExTaq was prepared by adding water sothat it has a volume of 50 μl, followed by layering 30 μl of mineraloil. For the reaction, a cycle of the three steps: denaturation (94° C.,30 seconds), annealing (55° C., 1 minute), and elongation (72° C., 1minute), were repeated 35 times using PCR Amplifier (ASTEC Co., Ltd.,PC-700). The above-described PCR amplified a DNA fragment having a sizeof about 1.0 kbp. After the reaction, the layered mineral oil wasextracted with 0.3 ml of hexane, and the hexane layer was removed. Thisprocedure was repeated three times, followed by reduction of thepressure for one minute, to thereby remove the mineral oil. From 50 μlof the thus-obtained reaction solution, PCR fragment was purified usingGENECLEAN (Bio101). Specifically, 300 μl of NaI solution included in thekit was added and mixed, and 10 μl of glass beads solution was added andmixed. The mixture was allowed to stand at 4° C. for 15 minutes andcentrifuged to precipitate glass beads to which the DNA fragment wasadsorbed, and the supernatant was removed. 500 μl of New wash solutionincluded in the kit was further added to suspend the glass beads, andthe mixture was centrifuged to remove the supernatant. The washingoperation using the New wash solution was repeated three times. Next,the glass beads were dried under reduced pressure, and after the drying,15 μl of sterilized water was added to suspend them. The mixture washeated to 55° C. for 15 minutes and centrifuged, to thereby yield 12 p.1 of a supernatant containing the DNA fragment.

An operation of inserting the purified DNA fragment into an expressionvector was performed as follows. Specifically, 0.5 μg of an expressionplasmid (pUC119: manufactured by Takara Shuzo Co., Ltd.)), 1 μl ofrestriction enzymes HindIII and EcoRI from Takara Shuzo Co., Ltd., and 2μl of 10×K buffer, which is a restriction enzyme buffer from TakaraShuzo Co., Ltd., were added to 10 μl of the DNA fragment solution, andsterilized water was added so that the mixture has a volume of 20 μl,followed by mixing. The reaction solution was allowed to react at 36° C.for 2 hours. After the reaction of the restriction enzymes, the DNAfragment and expression vector were isolated with GENECLEAN and ligatedwith each other. Specifically, 300 μl of the NaI solution included inthe kit was added to and mixed in 20 μl of the restriction enzymereaction solution, and 10 μl of the glass beads solution was addedthereto and mixed. The mixture was allowed to stand at 4° C. for 15minutes and then centrifuged to precipitate glass beads to which the DNAfragment and expression vector was adsorbed, and the supernatant wasremoved. Then, 500 μl of the New wash solution included in the kit wasadded to suspend the glass beads, and the suspension was centrifuged toremove the supernatant. The washing operation using the New washsolution was repeated three times. Next, the glass beads were driedunder reduced pressure, and after the drying, 15 μl of sterilized waterwas added to suspend them. The mixture was heated to 55° C. for 15minutes and centrifuged, to thereby yield 12 μl of a supernatantcontaining the DNA fragment and expression vector. The procedure removedsmall DNA fragments that have size of about 50 by or less and weregenerated by the restriction enzymes, to thereby yield the DNA fragmentof interest and expression vector.

10 μl of Takara Ligation kit-I solution (Takara Shuzo Co., Ltd.) wasadded to the 10 μl of the thus-prepared solution, and the mixture wasallowed to react at 16° C. for 1 hour. The solution was used totransform the competent cells (Takara Shuzo Co., Ltd.: DH5α).Specifically, 5 μl of a ligation reaction solution was added thereto andmixed in 60 p. 1 of a competent cell solution unfreezed at 4° C., andleft at 0° C. for 30 minutes, and then at 42° C. for 45 seconds and 0°C. for 2 minutes. 500 μl of SOC solution (2% bacto-tryptone, 0.5% yeastextract, 10 mM NaCl, 20 mM glucose, 10 mM MgSO₄, and 10 mM MgCl₂) wasadded thereto, followed by recovery culture at 36° C. for 1 hour. 100 p.1 of the culture solution was applied on LB agar medium (1%bacto-tryptone, 0.5% yeast extract, 1% NaCl, pH 7.0, 1.5% agar)containing 50 μg/ml ampicillin, 40 μg/ml X-gal(5-Bromo-4-Chloro-3-Indolyl-β-D-Galactoside), and 1 mM IPTG(thiogalactopyranoside). Culture was further performed at 37° C. for 16hours. The culture yielded Escherichia coli transformed by introducingthe above-described plasmid as white colonies, and the colonies wereselected. The thus-separated colonies of the transformed Escherichiacoli were cultured in LB liquid medium containing ampicillin (50 μg/ml).From the cultured cells of the transformed Escherichia coli, a plasmidDNA was separated and purified using a plasmid purification kit (QIAfilter Plasmid Midi Kit, QIAGEN). The thus-obtained plasmid DNA wasconfirmed to have a DNA fragment having a size of about 1.0 kbp, whichcorresponds to the ydgJ gene of interest.

Next, to confirm the scyllo-inositol dehydrogenase activity, themicrobial strains isolated as colonies were transferred to 100 ml of LBmedium (1% bacto-tryptone, 0.5% yeast extract, 1% NaCl, pH 7.0)containing 50 μg/ml ampicillin, and they were cultured at 36° C. for 7hours. 0.3 ml of 200 mM thiogalactopyranoside solution was added to theculture solution, and the cells were further cultured at 36° C. for 3hours. After completion of the culture, the cells were collected bycentrifugation and washed with physiological saline once. Then, thewashed cells were suspended in 3 ml of 0.6% Triton X-100 solution, andthe cells were disrupted by ultrasonic wave at 4° C. The solution wascentrifuged, and 2.8 ml of the supernatant (enzyme solution) was takenout. 1.2 g of ammonium sulfate was added to the supernatant to salt outproteins at 4° C. The salted-out proteins were collected bycentrifugation (15,000 rpm, 20 min), and the supernatant was removed.The precipitates were dissolved in 2.5 ml of 20 mM Tris buffer (pH 7.0),and the solution was centrifuged (15,000 rpm, 20 min) again. Thesupernatant was applied onto Sephadex G-25 column (Pharmacia K.K.: 14ml) equilibrated with 20 mM Tris buffer (pH 7.0). Elution was performedwith 20 mM Tris buffer (pH 7.0), and the eluate was desalted. Theprocedures yielded 3.5 ml of a crude enzyme solution of ydgJ geneproduct.

The scyllo-inositol dehydrogenase activity was measured as follows: 5 μlof a reaction solution (200 mM Tris buffer (pH 8.0), 2% of NADPH, and 1%of scyllo-inosose) and 5 μl of an enzyme solution were mixed and allowedto react at 36° C. for 30 minutes, and then 500 p. 1 of water wasimmediately added, followed by measurement of the absorbance at 340 nm.The decrease in the absorbance at 340 nm was measured based on a blankvalue for a test solution obtained by using water instead of the enzymesolution. The enzyme solution was diluted as required.

Meanwhile, the enzyme reaction product was measured as follows: 10 mg ofscyllo-inosose, 40 mg of NADPH, and 10 U of the enzyme were allowed toreact in 1.0 ml of 100 mM Tris buffer (pH 8.0) at 36° C. for 4 hours,and a heat treatment was performed at 80° C. for 10 min, followed bycooling. Then, 100 μl of a strong base cation exchange resin, 100 μl ofa strong acid anion exchange resin, and 10 mg of activated carbon wereadded, and the mixture was stirred and centrifuged. Then, thesupernatant was diluted 2-fold, and measurement was performed by HPLC(Shodex Asahipak NH2P-50 4E φ 4.6×250 mm: Shodex) using an RI detectorunder the condition of a column temperature of 40° C. and a mobile phaseflow rate of 1.5 ml (80% acetonitrile). As a result, the ydgJ geneproduct was found to have a high scyllo-inositol dehydrogenase activity,and 100% of the product was scyllo-inositol obtained by reduction, whilemyo-inositol that is an isomer thereof was not detected. As a result,the solution of a recombinant enzyme derived from the ydgJ gene wasfound to have a high scyllo-inositol dehydrogenase activity, and thegene product was scyllo-inositol dehydrogenase. Meanwhile, in theproduct obtained by the reduction reaction of scyllo-inosose, onlyscyllo-inositol was detected, and the enzyme was found tostereospecifically reduce scyllo-inosose into scyllo-inositol.Meanwhile, the sequence of the ydgJ gene is shown in SEQ ID NO: 1, andthe amino acid sequence corresponding thereto is shown in SEQ ID NO: 2.

Example 12 Isolation and Expression of a Homologous DNA Estimated fromHomology with Escherichia coli ydgJ Gene, and Properties thereof

From estimation of the three-dimensional structure of the ydgJ geneproduct of Escherichia coli based on the amino acid sequence, theproduct was estimated to belong to the family of glucose-fructoseoxidoreductases. The family also includes myo-inositol 2-dehydrogenase(EC 1.1.1.18), and the sequence involved in a NAD binding in the aminoacid sequence was found to have high homology. Moreover, identificationof the amino acid sequence of a site involved in the substrate bindingby X-ray structure analysis of glucose-fructose oxidoreductase andsearch of proteins that have partially the same amino acid sequence andare homologous with the ydgJ gene product revealed that there arehomologous proteins in many gram-negative bacteria and gram-positivebacteria.

From those bacteria, homologous DNAs were searched based on the homologyof the ydgJ gene of Escherichia coli. As a result, the ydgJ gene ofEscherichia coli was found to have homology with Atu4375 gene andAtu3234 gene in genome of Agrobacterium tumefacience C58 ATCC33970,BG14057 gene in genome of Bacillus subtilis 168 ATCC23857, Xcc3438 genein genome of Xanthomonas campestris pv. campestris ATCC33913, andAtu4375 gene and Atu3234 gene in genome of Agrobacterium sp. AB10121FERM P-17383 that is known as a microorganism having an ability todirectly convert myo-inositol into scyllo-inositol. Therefore, thoseDNAs were isolated and expressed.

To obtain the above-described candidate DNAs, the total genomes ofAgrobacterium tumefacience C58 ATCC33970, Bacillus subtilis 168ATCC23857, Xanthomonas campestris pv. campestris ATCC33913, andAgrobacterium sp. AB10121 FERM P-17383 to be used as templates wereextracted as follows. For Agrobacterium tumefacience C58, Xanthomonascampestris pv. campestris, and Agrobacterium sp. AB10121, one platinumloop of each of Agrobacterium tumefacience C58, Xanthomonas campestrispv. campestris, and Agrobacterium sp. AB10121 that had been cultured inLB slant medium (1% bacto-tryptone, 0.5% yeast extract, 1% NaCl, pH 7.0,1.5% agar) was inoculated in 100 ml of an LB flask medium (1%bacto-tryptone, 0.5% yeast extract, 1% NaCl, pH 7.0) and aerobicallycultured for 18 hours at 27° C. and collected. 15 ml of Saline-EDTAsolution (0.15 M NaCl, 0.1 M EDTA, pH 8.0) and 50 mg of lysozyme wereadded to the cell pellets to suspend it, followed by reaction at 37° C.for 2 hours. After the treatment, 0.5 ml of 25% SDS solution was addedto the solution to completely lyse the cells, and 3 ml of phenol wasadded to denature proteins. Thereafter, the solution was centrifuged,and the supernatant was taken out. 20 ml of 2-propanol was added to thesolution to yield crude genomic DNA. The yielded crude genomic DNA wasprecipitated by centrifugation, and the supernatant was removed,followed by drying under reduced pressure. Thereafter, dried crudegenomic DNA was further dissolved in 3 ml of TE solution (10 mMTris-HCl, 1 mM EDTA, pH 8.0), and then 0.01 mg of RNAase was addedthereto and allowed to react at 36° C. for 2 hours to degrade RNA.Moreover, 0.01 mg of proteinase K was added and allowed to react at 36°C. for 2 hours to degrade proteins. Next, 1 ml of phenol-chloroformmixed solution (1:1) was added, and the mixture was slowly stirred todenature RNAase and proteinase K. The mixture was separated into twophases by centrifugation, and the upper layer (aqueous phase) was takenout and was adjusted to pH 5.2 by adding 0.3 ml of 3 M sodium acetatesolution. 3 ml of 2-propanol was added to the solution to yield genomicDNA. The obtained genomic DNA was precipitated by centrifugation, andthe supernatant was removed, followed by drying under reduced pressure.The dried genomic DNA was dissolved in 3 ml of TE solution, and theprocess from the operation of adding 1 ml of the phenol-chlorofoim mixedsolution (1:1) to the operation of dissolving DNA in 3 ml of the TEsolution was repeated again, followed by centrifugation. Then, in thesame way as above, equal volume of 2-propanol were added to thesupernatant at pH 5.2, to thereby prepare solutions of genomic DNAs ofAgrobacterium tumefacience C58, Xanthomonas campestris pv. campestris,and Agrobacterium sp. AB10121, respectively. The thus-obtained genomicDNAs were used as template DNA solutions for PCR.

One platinum loop of Bacillus subtilis 168 ATCC23857 that had beencultured in an LB slant medium (1% bacto-tryptone, a 0.5% yeast extract,1% NaCl, pH 7.0, 1.5% agar) was inoculated in 100 ml of LB flask medium(1% bacto-tryptone, 0.5% yeast extract, 1% NaCl, pH 7.0) and aerobicallycultured at 36° C. for 18 hours. Then, 1 ml of the medium was added to100 ml of an LB flask medium prepared as above, and the microorganismswere cultured for 4 hours, followed by collection. After the collection,the total genome was extracted in the same manner as the method used forAgrobacterium.

Next, PCR was performed using primers having the following sequences forcloning of Atu4375 gene and Atu3234 gene in the genome of Agrobacteriumtumefacience C58, Atu4375 gene and Atu3234 gene in the genome ofAgrobacterium sp. AB10121, and Xcc3438 gene in the genome of Xanthomonascampestris pv. campestris (all including RBS), for exnressing them asrecombinant enzymes.

SEQ ID No. 17: Atu4375-F 5′-ggcggatcctttgaaagggatagtcatgtcct-3′ SEQ IDNo. 18: Atu4375-R 5′-attggaagcttcgattggctgcgacctag-3′ SEQ ID No. 19:Atu3234-F 5′-ttgggatcctttcaggggaaatattatggc-3′ SEQ ID No. 20: Atu3234-R5′-gccgcaagcttgttttacagatcac-3′ SEQ ID No. 23: Xcc3438-F5′-tcggaattcgcgttgcggtgaatcgtittcaatg-3′ SEQ ID No. 24: Xcc3438-R5′-ataagaagcttgctcagtcgctgctgttgccttc-3′

PCR was performed using primers having the following sequences forcloning of BG14057 gene in the genome of Bacillus subtilis 168(including RBS) for expressing it as a recombinant enzyme. The “a” atposition 10 from the 5′-terminus is originally “t” but is altered to “a”for expression in Escherichia coli.

SEQ ID No. 21: BG14057-F 5′-aggaattcgatgataacgcttttaaaggggagaa-3′ SEQ IDNo. 22: BG14057-R 5′-tttctgcagtttagtgctccagcataatggttcg-3′

For PCR, Ex taq reaction solution from Takara Shuzo Co., Ltd. was used,and a solution having a composition of 5 ml of 10× Takara ExTaq Buffer,4 μl of a dNTP mixture, 30 ng of a template DNA, 1 μl of 10 μM primersolution, and 0.5 ml of Takara ExTaq was prepared by adding water sothat it has a volume of 50 μl, followed by layering 30 ml of mineraloil. For reaction, a cycle of three steps: denaturation (94° C., 30seconds), annealing (52° C., 55° C., or 58° C. (See Table 3), 1 minute),and elongation (72° C., 1 minute), were repeated 35 times using PCRAmplifier (ASTEC Co., Ltd., PC-700). The above-described PCR amplified aDNA fragment having a size of about 1.1 kbp.

TABLE 3 List of annealing temperature Annealing Target genes temperatureFor Atu4375 gene of Agrobacterium tumefacience C58 ATCC33970 55° C. ForAtu4375 gene of Agrobacterium AB10121 FERM P-17383 55° C. For Atu3234gene of Agrobacterium tumefacience C58 ATCC33970 52° C. For Atu3234 geneof Agrobacterium AB10121 FERM P-17383 52° C. For BG14057 gene ofBacillus subtilis 168 ATCC23857 55° C. For Xcc3438 gene of Xanthomonascampestris pv. Campestris ATCC33913 58° C.

After the reaction, the layered mineral oil was extracted with 0.3 ml ofhexane, and the hexane layer was removed. This procedure was repeatedthree times, followed by reduction of the pressure for one minute, tothereby remove the mineral oil. From 50 μl of the thus-obtained reactionsolution, PCR fragments were purified using GENECLEAN (Bio101).Specifically, 300 μl of the NaI solution included in the kit was addedand mixed, and 10 μl of a glass beads solution was added and mixed. Themixture was allowed to stand at 4° C. for 15 minutes and centrifuged toprecipitate glass beads to which the DNA fragments were adsorbed, andthe supernatant was removed. 500 μl of New wash solution included in thekit was further added to suspend the glass beads, and the mixture wascentrifuged to remove the supernatant. The washing operation using theNew wash solution was repeated three times. Next, the glass beads weredried under reduced pressure, and after the drying, 15 μl of sterilizedwater was added to suspend them. The mixture was heated to 55° C. for 15minutes and centrifuged, to thereby yield 12 μl of a supernatantcontaining DNA fragments.

An operation of inserting the purified DNA fragment into an expressionvector was performed in each of the combinations as follows.Specifically, 0.5 μg of an expression plasmid (pUC118: manufactured byTakara Shuzo Co., Ltd.)), 1 μl of two kinds of restriction enzymes fromTakara Shuzo Co., Ltd., and 2 μl of 10×K buffer, which is a restrictionenzyme buffer solution from Takara Shuzo Co., Ltd., were added to 10 μlof the DNA fragment solution, and sterilized water was added so that themixture has a volume of 20 μl, followed by mixing. The reaction solutionwas allowed to react at 36° C. for 2 hours. As the Atu3234 gene of theAB10121 strain contains a HindIII site, the treatment with restrictionenzymes was not conducted, and after isolation, ligated to a pT7Bluevector (manufactured by Novagen).

TABLE 4 List of expression plasmids and used restriction enzymesExpression Target genes plasmids Used restriction enzymes Atu4375 geneof A. tume. C58 pUC118 BamH I, Hind III/K buffer Atu4375 gene of AB10121pUC118 BamH I, Hind III/K buffer Atu3234 gene of A. tume. C58 pUC118BamH I, Hind III/K buffer Atu3234 gene of AB10121 pT7Blue Not usedBG14057 gene of B. sub. 168 pUC118 EcoR I, Pst I/H buffer Xcc3438 geneof X. camp. pUC118 EcoR I, Hind III/K buffer

After the reaction of the restriction enzymes, DNA fragment andexpression vector were isolated with GENECLEAN and ligated with eachother. Specifically, 300 μl of the NaI solution included in the kit wasadded to 20 μl of the restriction enzyme reaction solution and mixed,and 10 μl of the glass beads solution was added and mixed. The mixturewas allowed to stand at 4° C. for 15 minutes and then centrifuged toprecipitate glass beads to which the DNA fragment and expression vectorwere adsorbed, and the supernatant was removed. Moreover, 500 p. 1 ofthe New wash solution included in the kit was added to suspend the glassbeads, and the suspension was centrifuged to remove the supernatant. Thewashing operation using the New wash solution was repeated three times.Next, the glass beads were dried under reduced pressure, and after thedrying, 15 μl of sterilized water was added to suspend them. The mixturewas heated to 55° C. for 15 minutes and centrifuged, to thereby yield 12μl of a supernatant containing DNA fragment and expression vector. Theprocedure removed small DNA fragments generated by the restrictionenzymes that have sizes of about 50 by or less, to thereby yield a DNAfragment of interest and expression vector.

10 μl of Takara Ligation kit-I solution (Takara Shuzo Co., Ltd.) wasadded to 10 p. 1 of the thus-prepared solution, and the mixture wasallowed to react at 16° C. for 1 hour. The solution was used totransform competent cells (Takara Shuzo Co., Ltd.: DH5α). Specifically,5 μl of a ligation reaction solution was added to 60 μl of a competentcell solution unfreezed at 4° C. and mixed, and left for at 0° C. 30minutes, then at 42° C. for 45 seconds and 0° C. for 2 minutes. 500 μlof SOC solution (2% bacto-tryptone, 0.5% yeast extract, 10 mM NaCl, 20mM glucose, 10 mM MgSO₄, and 10 mM MgCl₂) was added thereto, followed byrecovery culture at 36° C. for 1 hour. 100 μl of the culture solutionwas applied to an LB agar medium (1% bacto-tryptone, 0.5% yeast extract,1% NaCl, pH 7.0, 1.5% agar) containing 50 μg/ml ampicillin, 40 μg/mlX-gal (5-Bromo-4-Chloro-3-Indolyl-β-D-Galactoside), and 1 mM IPTG(thiogalactopyranoside). Culture was further performed at 37° C. for 16hours. The culture yielded Escherichia coli transformed by introducingthe above-described plasmid as white colonies, and the colonies wereselected. The thus-separated colonies of transformed Escherichia coliwere cultured in an LB liquid medium containing ampicillin (50 μg/ml).From the cultured cells of the transformed Escherichia coli, plasmid DNAwas separated and purified using a plasmid purification kit (QIA filterPlasmid Midi Kit, QIAGEN). The thus-obtained plasmid DNA was confirmedto each have a DNA fragment having a size of about 1.0 to 1.1 kbp, whichcorresponds to the DNA of interest.

Next, to confirm the scyllo-inositol dehydrogenase activity, themicrobial strains isolated as colonies were transferred to 100 ml of anLB medium (1% bacto-tryptone, 0.5% yeast extract, 1% NaCl, pH 7.0)containing 50 μg/ml ampicillin, and they were cultured at 36° C. for 7hours. 0.3 ml of 200 mM thiogalactopyranoside solution was added to theculture solution, and the cells were further cultured at 36° C. for 3hours. After completion of the culture, the cells were collected bycentrifugation and washed with physiological saline once. Then, thewashed cells were suspended in 3 ml of 0.6% Triton X-100 solution, andthe cells were disrupted by ultrasonic wave at 4° C. The solution wascentrifuged, and 2.8 ml of the supernatant (enzyme solution) was takenout. 1.2 g of ammonium sulfate was added to the supernatant to salt outproteins at 4° C. The salted-out proteins were collected bycentrifugation, and the supernatant was removed. The precipitates weredissolved in 2.5 ml of 20 mM Tris buffer (pH 7.0), and the solution wascentrifuged again. The supernatant was applied onto a Sephadex G-25column (14 ml) equilibrated with 20 mM Tris buffer (pH 7.0). Elution wasperformed with 20 mM Tris buffer solution (pH 7.0), and the eluate wasdesalted. The procedures yielded 3.5 ml of a crude enzyme solution ofydgJ gene product.

The scyllo-inositol dehydrogenase activity was measured as follows: 5 μlof a reaction solution (200 mM Tris buffer (pH 8.0), 2% of NADPH, and 1%of scyllo-inosose) and 5 μl of an enzyme solution were mixed and allowedto react at 36° C. for 30 minutes, and then 500 μl of water wasimmediately added, followed by measurement of the absorbance at 340 nm.The decrease in the absorbance at 340 nm was measured based on a blankvalue for a test solution obtained by using water instead of the enzymesolution. The enzyme solution was diluted as required.

Meanwhile, enzyme reaction product was measured as follows: 10 mg ofscyllo-inosose, 40 mg of NADPH, and 10 U of the enzyme were allowed toreact in 1.0 ml of 100 mM Tris buffer (pH 8.0) at 36° C. for 4 hours,and a heat treatment was performed at 80° C. for 10 min, followed bycooling. Then, 100 μl of a strong base cation exchange resin, 100 μl ofa strong acid anion exchange resin, and 10 mg of activated carbon wereadded, and the mixture was stirred and centrifuged. Then, thesupernatant was diluted 2-fold, and measurement was performed by HPLC(Shodex Asahipak NH2P-50 4E φ 4.6×250 mm: Shodex) using an RI detectorunder conditions of a column temperature of 40° C. and a mobile phaseflow rate of 1.5 ml (80% acetonitrile). As a result, the Atu4375 geneproduct and the Atu3234 gene product of Agrobacterium tumefacience C58,the BG14057 gene product of Bacillus subtilis 168, the Xcc3438 geneproduct of Xanthomonas campestris pv. campestris, and the Atu4375 geneproduct, and the Atu3234 gene product of AB10121 strain were found tohave a high enzyme activity, and 100% of the product was scyllo-inositolobtained by the reduction, while myo-inositol that is an isomer thereofwas not detected. As a result, recombinant enzymes derived from theabove-mentioned genes were found to have a high scyllo-inositoldehydrogenase activity, and the gene products were scyllo-inositoldehydrogenase. Meanwhile, in the products obtained by the reductionreaction of scyllo-inosose, only scyllo-inositol was detected, and theenzymes were found to stereospecifically reduce scyllo-inosose intoscyllo-inositol.

The sequence of Atu4375 gene derived from Agrobacterium tumefacience C58is shown in SEQ ID NO: 3, the corresponding amino acid sequence is shownin SEQ ID NO: 4, the sequence of Atu3234 gene is shown in SEQ ID NO: 5,and the corresponding amino acid sequence is shown in SEQ ID NO: 6.Meanwhile, the sequence of BG14057 gene derived from Bacillus subtilis168 is shown in SEQ ID NO: 7, the corresponding amino acid sequence isshown in SEQ ID NO: 8, the sequence of Atu4375 gene derived from AB10121is shown in SEQ ID NO: 9, the corresponding amino acid sequence is shownin SEQ ID NO: 10, the sequence of Atu3234 gene is shown in SEQ ID NO:11, the corresponding amino acid sequence is shown in SEQ ID NO: 12, thesequence of Xcc3438 gene derived from Xanthomonas campestris pv.campestris is shown in SEQ ID NO: 13, and the corresponding amino acidsequence is shown in SEQ ID NO: 14. Meanwhile, from the results ofnucleotide sequence analysis of a plasmid including Atu4375 gene andAtu3234 gene of AB10121 (Hokkaido System Science Co., Ltd.), thehomology between Atu4375 gene of Agrobacterium tumefacience C58 andAtu4375 gene of AB10121 was 89% for the nucleotide sequence, and 96% forthe amino acid sequence, while the homology between Atu3234 gene ofAgrobacterium tumefacience C58 and Atu3234 gene of AB10121 was 87% forthe nucleotide sequence and 95% for the amino acid sequence.

Example 13 Isolation of the DNA Encoding SIDH1 which is Produced fromAcetobacter sp. AB10281 FERM BP-10119>

Of the enzymes purified, an enzyme solution containing SIDH1 was appliedonto a PVDF membrane (Immobilon PSQ: manufactured by MilliporeCorporation), and absorbed to the PVDF membrane. The PVDF membrane wastaken out, stained with Coomassie brilliant blue stain (Rapid CBB KANTO:manufactured by KANTO CHEMICAL CO., INC.), decolorized, and dried,followed by analysis with an N-terminal amino acid analyzer(manufactured by Hewlett-Packard Development Company). As a result, fromthe N-terminal, a sequence:Met-Lys-Arg-Lys-Leu-Arg-Ile-Gly-Leu-Ile-Gly-Ser-Gly-Phe-Met-Gly-Arg-Thr-His-Ala-Phe-Gly-Tyr-Serwas identified. Then a DNA sequence encoding the amino acid sequence waspredicted, and the following two kinds of primers were constructed.

SEQ ID NO: 29: SIDH1-F1 atgaarcgnaarytncgiatyggyytiatygg SEQ ID NO: 30:SIDH1-F2 ggyttyatgggycgnacicaygcittyggyta

Next, based on the nucleotide sequences of various scyllo-inositoldehydrogenases obtained in Examples 10 to 12, there were prepared thefollowing two primers including highly consensus regions within thesequences.

SEQ ID NO: 31: SIDH1-B1 ggyttrtcrmmgayracrtgrstrcc SEQ ID NO: 32:SIDH1-B2 artgwrirtgrttgggigt

Meanwhile, as genomic DNA of AB10281 to be used as a template, asolution of genomic DNA of AB10281 was prepared using the cell pellet(wet weight about 400 mg) prepared in Example 9 in the same way as themethod of preparing DNA described in Example 12.

For PCR, Ex taq reaction solution from Takara Shuzo Co., Ltd. was used,and a solution having a composition of 5 μl of 10× Takara ExTaq Buffer,4 μl of dNTP mixture, 30 ng of a template DNA, 1 μl of each 10 μM primersolution (SIDH1-F1 and SIDH1-B1), and 0.5 μl of Takara ExTaq wasprepared by adding water so that it has a volume of 50 μl, followed bylayering 30 μl of mineral oil. For reaction, PCR Amplifier (ASTEC Co.,Ltd., PC-700) was used, and a cycle of three steps: denaturation (94°C., 30 seconds), annealing (50° C., 30 seconds), and elongation (72° C.,1 minute), was repeated 35 times. Electrophoresis revealed that theabove-described PCR amplified a DNA fragment having a size of about 0.3kbp. Moreover, the band at the position of 0.3 kbp was excised from thegel, a part of a homogenized gel solution was used as a template (2 μl),and there was prepared a reaction solution having the same compositionas described above except that a combination of primers was changed toSIDH1-F2 and SIDH1-B2. For reaction, PCR Amplifier (ASTEC Co., Ltd.,PC-700) was used, and a cycle of three steps: denaturation (94° C., 30seconds), annealing (46° C., 30 seconds), and elongation (72° C., 1minute), was repeated 35 times. Electrophoresis revealed that the PCRamplified a DNA fragment having a size of about 0.25 kbp.

The DNA fragment having a size of about 0.25 kbp was excised from thegel, and the PCR fragment was purified using GENECLEAN (manufactured byBio101). Specifically, in the same way as the purification methoddescribed in Example 12 except that the gel was dissolved in NaIsolution, 12 μl of a solution of the DNA fragment was obtained.

Next, the purified DNA fragment was ligated to a pT7Blue vector.Specifically, 0.5 μg of the pT7Blue vector and 10 μl of a TakaraLigation kit-I solution (Takara Shuzo Co., Ltd.) were added to 10 μl ofthe DNA fragment solution, and the mixture was allowed to react at 16°C. for 1 hour. The solution was used to transform competent cells (DH5α,Takara Shuzo Co., Ltd.). The operation of transformation and isolationof a plasmid from transformants were performed in the same way asExample 12.

The obtained plasmid was subjected to nucleotide sequence analysis(Hokkaido System Science Co., Ltd.) using universal primers (R-20mer andU-19mer), and about one third of the former half region of thenucleotide sequence of a gene encoding the enzyme was revealed.

Then, to determine the full length of the nucleotide sequence, genomicDNA of AB10281 was completely digested with a restriction enzyme BamHI,and the resultant DNA fragment solution was purified using GENECLEAN(manufactured by Bio101), followed by self-ligation of the fragment.Specifically, 10 μl of Takara Ligation kit-I solution (Takara Shuzo Co.,Ltd.) was added to 10 μl of the DNA fragment solution, and the mixturewas allowed to react at 16° C. for 1 hour. After the reaction, the DNAwas purified using GENECLEAN (manufactured by Bio101), and the solutionwas used as a template DNA solution for inverse PCR.

Next, based on the revealed about one third of the former half region ofthe nucleotide sequence, the following three primers were prepared, andinverse PCR was performed.

SEQ ID No: 33: SIDH1-INV-F gctcgtcaacgatcctgaaattgat SEQ ID No: 34:SIDH1-INV-B ttcgctgcagcttcatcggaaatat SEQ ID No: 35: SIDH1-INVF3cccttcaatttccgggcgggt

For inverse PCR, Ex taq reaction solution from Takara Shuzo Co., Ltd.was used, and a solution having a composition of 5 μl of 10× TakaraExTaq Buffer, 4 μl of dNTP mixture, 30 ng of a template DNA, 1 μl ofeach 10 μM primer solution (a combination of SIDH1-INV-F andSIDH1-INV-B, or a combination of SIDH1-INV-F and SIDH1-INV3), and 0.5 μlof Takara ExTaq was prepared by adding water so that it has a volume of50 followed by layering 30 μl of mineral oil. For reaction, PCRAmplifier (ASTEC Co., Ltd., PC-700) was used, and a cycle of threesteps: denaturation (94° C., 30 seconds), annealing (50° C., 1 minute),and elongation (72° C., 2 minutes), was repeated 35 times.Electrophoresis revealed that the above-described PCR amplified DNAfragments having sizes of about 2.7 kbp and about 1.8 kbp. Then, thebands at the positions of about 2.7 kbp and about 1.8 kbp were excisedfrom the gel, and the PCR fragments were purified using GENECLEAN(manufactured by Bio101), to thereby yield 10 μl of DNA fragmentsolutions. The resultant two DNA fragments were subjected to nucleotidesequence analysis (Hokkaido System Science Co., Ltd.) using the primersused in PCR to determine the entire nucleotide sequence of the genesencoding the enzyme.

Next, PCR was performed using primers having the following sequences forcloning SIDH1 gene (including an RBS site) derived from AB10281 strainfor expressing it as a recombinant enzyme.

SEQ ID No: 36: 281SIDH1-F gctggatcccgcccttattgtgaata SEQ ID No: 37:281SIDH1-R tatgaattcgttatgccttctcatgctgtcg

For PCR, Ex taq reaction solution from Takara Shuzo Co., Ltd. was used,and a solution having a composition of 5 μl of 10× Takara ExTaq Buffer,4 μl of dNTP mixture, 30 ng of a template DNA, 1 μl of each 10 μM primersolution, and 0.5 μl of Takara ExTaq was prepared by adding water sothat it has a volume of 50 μl, followed by layering 30 μl of mineraloil. For reaction, a cycle of three steps: denaturation (94° C., 30seconds), annealing (55° C., 1 minute), and elongation (72° C., 1minute), was repeated 35 times using PCR Amplifier (ASTEC Co., Ltd.,PC-700). The above-described PCR amplified a DNA fragment having a sizeof about 1.2 kbp.

The DNA fragment having a size of about 1.2 kbp was purified usingGENECLEAN (manufactured by Bio101), to thereby yield 12 μl of a DNAfragment solution. For a procedure to insert the DNA fragment into anexpression vector, 0.5 μg of an expression plasmid (pUC119), 1 μl ofrestriction enzymes (BamHI, EcoRI) from Takara Shuzo Co., Ltd., and 2 μlof 10×K buffer for restriction enzymes from Takara Shuzo Co., Ltd. wereadded to 10 μl of the DNA fragment solution, and sterilized water wasadded so as to have a volume of 20 μl, followed by mixing. The reactionsolution was allowed to react at 36° C. for 2 hours.

Collection of the DNA fragment from the reaction solution, purification,ligation reaction, and transformation of competent cells (DH5α, TakaraShuzo Co., Ltd.) were performed in the same way as Example 12. Moreover,the expression of scyllo-inositol dehydrogenase and the activitymeasurement were performed in the same way as Example 12, and the geneproduct was identified as scyllo-inositol dehydrogenase.

As a result, the nucleotide sequence of a gene encoding SIDH1 derivedfrom AB10281 strain is shown in SEQ ID NO: 27, while its amino acidsequence is shown in SEQ ID NO: 28.

Example 14 Study on Properties of the Enzyme of the Present Invention,Various Kinds of Scyllo-inositol Dehydrogenase

The following method was performed to study the enzymatic properties ofSIDH1, SIDH2, and SIDH3 derived from AB10281 strain, which had beenobtained by enzyme purification in Example 9; the ydgJ gene productderived from Escherichia coli, which had been obtained as a recombinantenzyme in Example 11; and the Atu4375 and Atu3234 gene products ofAgrobacterium tumefacience C58, the BG14057 gene product of Bacillussubtilis 168, the Xcc3438 gene product of Xanthomonas campestris pv.campestris, and the Atu4375 and Atu3234 gene products of the AB10121strain, all of which had been obtained as recombinant enzymes in Example12, and the results are shown in Table 5.

Table 6 shows the homology in the amino acid sequence. The homology ofonly the amino acids common in all the sequences was low (about 5%), butthe homology including amino acids having similar properties was highparticularly in NAD or NADP binding domains in about 30% of the sequenceof the N-terminal. Moreover, the lysine-proline sequence located forwardthe center of the sequence involved in binding of nicotinamide, which isan oxidoreduction reaction site of NAD or NADP, was highly conserved.Asparagine at the 27th position toward the C-terminal from thelysine-proline sequence and the aspartic acid-(3 amino acids)-histidinesequence near the center of the sequence were also highly conserved, sothat they are considered as important sequences involved in thesubstrate binding from the estimated three-dimensional structures. Inthe table, the common sequences are represented by the symbol “*”, theamino acids having similar properties are represented by the symbol “:”or the symbol “.”, and Asparagine at the 27th position toward theC-terminal from the lysine-proline sequence and the aspartic acid-(3amino acids)-histidine sequence near the center of the sequence arerepresented by hatching.

In the comparison of molecular weights, the molecular weight of theenzyme of the present invention derived from AB10281 was calculatedbased on the results of SDS-PAGE using a molecular weight marker(prestain standard (broad range type): manufactured by Bio-RadLaboratories, Inc.), while the molecular weights of the other enzymes ofthe present invention were estimated from the full lengths of the genes.As a result, the molecular weights of the enzymes of the presentinvention (SIDH1, SIDH2, and SIDH3) derived from AB10281, which had beenobtained by enzyme purification, were 46 k Dalton, 42 k Dalton, and 40 kDalton, respectively. Meanwhile, the molecular weight of the enzyme ofthe present invention derived from ydgJ gene of Escherichia coli K-12,which had been obtained as a recombinant enzyme in Example 11, was 38.2k Dalton; the molecular weights of the enzymes of the present inventionderived from Atu4375 gene and Atu3234 gene of Agrobacterium tumefacienceC58, which had been obtained as recombinant enzymes in Example 12, were41.3 k Dalton and 42.4 k Dalton, respectively; the molecular weights ofthe enzymes of the present invention derived from BG14057 gene ofBacillus subtilis 168, Xcc3438 gene of Xanthomonas campestris pv.campestris, and Atu4375 gene and Atu3234 gene of AB10121 were 40.1 kDalton, 38.5 k Dalton, 41.4 k Dalton, and 42.5 k Dalton, respectively.That is, the enzymes of the present invention, scyllo-inositoldehydrogenase, were found to have molecular weight of 38 to 46 k Dalton.

Association properties of the enzyme of the present invention weredetermined by: measuring the activity of fractions obtained byfractionation using a gel filtration column (Tosoh Corporation:2000SWXL); calculating the molecular weight from the correspondingmolecular weight fractions; and dividing the calculated values by themolecular weights of the enzyme. The resultant values were representedas integers. As a result, the enzyme of the present invention,scyllo-inositol dehydrogenases, was found to have molecular weight of 80k to 110 k Dalton, and was considered to form dimmer or trimer in theundenatured conditions.

Coenzyme selectivity of the enzyme of the present invention wasdetermined by: mixing 5 μl of a reaction solution (200 mM Tris buffer(pH 8.0), 2% of NADPH or NADH, and 1% of scyllo-inosose) with 5 μl of anenzyme solution; and allowing the mixture to react at 36° C. for 30 min.After the reaction, 500 μl of water was immediately added thereto, andthe absorbance at 340 nm was measured. From the blank value of a testsolution prepared by using water instead of the enzyme solution, thedecrease in the absorbance at 340 nm was measured. The enzyme solutionwas diluted as required. The results indicate that scyllo-inositoldehydrogenases of the enzymes of the present invention were able to useboth NADPH and NADH as coenzymes, but have the coenzyme relativeactivity as shown in Table 5. It was revealed that many enzymes havehigh reactivity with NADPH.

The optimum pH of the enzymes of the present invention was determinedby: mixing 5 μl of a reaction solution (200 mM phosphate buffer (pH 5.0to 9.0), 2% of NADPH, and 1% of scyllo-inosose) with 5 μl of an enzymesolution; and allowing the mixture to react at 36° C. for 30 min. Afterthe reaction, 500 μl of water was immediately added thereto, and theabsorbance at 340 nm was measured. From the blank value of a testsolution prepared by using water instead of the enzyme solution, thedecrease in the absorbance at 340 nm was measured. The enzyme solutionwas diluted as required. As a result, the enzymes of the presentinvention, scyllo-inositol dehydrogenase, were found to have optimum pHas shown in Table 5. That is, the enzymes of the present invention werefound to react in the wide range of pH 5 to 9. Meanwhile, it was alsorevealed that there are enzymes having maximum activity at the acidicside (about pH 6), enzymes having maximum activity at the neutral region(about pH 6.5 to 7.5), and enzymes having maximum activity at thealkaline side (about pH 7.5 to 9).

The thermostability of the enzymes of the present invention wasdetermined by: treating an enzyme solution at the predeterminedtemperature for 10 min; cooling the solution; mixing the enzyme solutionwith 5 μl of a reaction solution (200 mM phosphate buffer (pH 5.0 to9.0), 2% of NADPH, and 1% of scyllo-inosose); and allowing the mixtureto react at 36° C. for 30 min. Thereafter, 500 μl of water wasimmediately added thereto, and the absorbance at 340 nm was measured.From the blank value of a test solution prepared by using water insteadof the enzyme solution, the decrease in the absorbance at 340 nm wasmeasured. The enzyme solution was diluted as required. The activity of agroup treated at 20° C. for 10 min was defined as 100%, and the relativeactivities were compared. As a result, the thermostability of theenzymes of the present invention, scyllo-inositol dehydrogenase, wasfound to vary depending on enzymes as shown in Table 5, and thestability was found to vary in the range of 40 to 60° C. depending onthe enzymes.

The effects of heavy metal on the enzymes of the present invention weredetermined by: mixing 5 μl of a reaction solution (200 mM Tris buffer(pH 8.0), 2% of NADPH, 1% of scyllo-inosose, and 2 mM of a heavy metal)with 5 μl of an enzyme solution; and allowing the mixture to react at36° C. for 30 min. After the reaction, 500 μl of water was immediatelyadded thereto, and the absorbance at 340 nm was measured. From the blankvalue of a test solution prepared by using water instead of the enzymesolution, the decrease in the absorbance at 340 nm was measured. Theenzyme solution was diluted as required. CaCl₂, CoCl₂, ZnSO₄, MgSO₄,SnCl₂, NiCl₂, and MnSO₄ were used as metallic salts. The activity of theno addition group was defined as 100%, and the relative activities werecompared. As a result, as shown in Table 5, the enzymes of the presentinvention, scyllo-inositol dehydrogenase, were activated at least in thepresence of Co²⁺ ion and inhibited in the presence of Sn²⁺ ion. Most ofthe enzymes were inhibited in the presence of Zn²⁺ ion, in contrast, theenzyme derived from Bacillus subtilis 168 was activated in the presenceof Zn²⁺ ion.

The Km values of the enzymes of the present invention for scyllo-inososewere determined by: mixing 5 μl of a reaction solution (200 mM Trisbuffer (pH 8.0), 2% of NADPH, and 0.001 to 2.5% of scyllo-inosose) with5 μl of an enzyme solution; and allowing the mixture to react at 36° C.for 30 min. After the reaction, 500 μl of water was immediately addedthereto, and the absorbance at 340 nm was measured. From the blank valueof a test solution prepared by using water instead of the enzymesolution, the decrease in the absorbance at 340 nm was measured. Theenzyme solution was diluted as required. The Km values were calculatedby the reciprocal plot. As a result, as shown in Table 5, the enzymes ofthe present invention, scyllo-inositol dehydrogenase, were found to haveKm values in the range of 2.6 to 12.6 mM.

The substrate specificity of the enzymes of the present invention wasdetermined by measuring the relative activity of oxidation with respectto the reactivity to scyllo-inositol. The inositol isomers includescyllo-inositol (SI), myo-inositol (MI), D-chiro-inositol (DCI),L-chiro-inositol (LCI), epi-inositol (EI), muco-inositol (MuI),allo-inositol (AI), and neo-inositol (NI). Table 5 shows the group ofisomers to which the enzyme shows a relative activity of not less than70%, a group of isomers to which the enzyme shows a relative activity ofless than 70% and not less than 20%, and a group of isomers to which theenzyme shows a relative activity of less than 20%.

The substrate specificity was determined by: mixing 50 μl of a reactionsolution (1% inositol isomers (or 0.4%: only for neo-inositol), 200 mMTris buffer (pH 8.0), 0.002% NADP⁺, 0.002% diaphorase, and 0.01%nitrotetrazolium blue) with 50 μl of an enzyme solution; and measuringthe increase in the absorbance at 545 nm at 25° C. every three minutesusing a microplate reader. The reaction rate was calculated from theabsorbance increments at the respective times. As a result, thesubstrate specificity was found to vary slightly depending on the kindsof the enzymes, and from the correlation with the structures of theinositol isomers, it was also found that those enzymes have at leastscyllo-inositol dehydrogenase activity and myo-inositol dehydrogenaseactivity.

TABLE 5 List of properties of scyllo-inositol dehydrogenase Strains E.coli Acetobacter sp. Bacillus sub. Strain K12 AB10281 168 strain(ATCC10798) (FERM P-18868) (ATCC23857) Gene name or enzyme name ydgJgene SIDH1 SIDH2 SIDH3 BG14057 gene Molecular weight kDalton 38.2k46k(SDS-PAGE) 42k(SDS-PAGE) 40k(SDS-PAGE) 40.1k Association propertyDimer Trimer Dimer Dimer Dimer Thermostability Stable at 45° C. orStable at 45° C. or Stable at 60° C. or Stable at 60° C. or Stable at40° C. or less less less less less Coenzyme relative activity NADPH:NADH= NADPH:NADH = NADPH:NADH = NADPH:NADH = NADPH:NADH = 100:9 100:112100:1 100:3 100:52 Optimum pH pH7.5-9.0 pH5.5-6.5 pH5.5-6.5 pH5.5-6.5pH7.0-8.5 Heavy metal effects: activation Co Co Co Co, Mn Co, Mn, ZnHeavy metal effects: strong inhibition Sn, Zn Sn, Zn Sn, Zn Sn, Zn SnReduction reaction product Only SIS → SI Only SIS → SI Only SIS → SIOnly SIS → SI Only SIS → SI Km values for scyllo-inosose 3.9 mM 7.6 mM10.6 mM 12.6 mM 3.5 mM Substrate specific Relative SI, MI, DCI SI SI SISI, MI activity (70% or more) Substrate specific Relative LCI, EI, MuIMI MI MI DCI, EI, AI, NI activity (20 to 70%) Substrate specificRelative AI, NI DCI, LCI, EI, DCI, LCI, EI, DCI, LCI, EI, LCI, MuIactivity (less than 20%) MuI, AI, NI MuI, AI, NI MuI, AI, NI StrainsXanthomonas Agrobacterium tumefacience Agrobacterium sp. campestrisStrain C58 AB10121 pv. Campestris (ATCC33970) (FERM P-17383) (ATCC33913)Gene name or enzyme name Atu4375 gene Atu3234 gene Atu4375 gene Atu3234gene Xcc3438 gene kDalton 41.3k 42.4k 41.4k 42.5k 38.5k Associationproperty Dimer Dimer Dimer Dimer Dimer Thermostability Stable at 50° C.Stable at 40° C. or Stable at 50° C. or Stable at 40° C. or Stable at40° C. or less less less less or less Coenzyme relative activityNADPH:NADH = NADPH:NADH = NADPH:NADH = NADPH:NADH = NADPH:NADH = 100:9100:18 100:6 100:20 100:34 Optimum pH pH6.5-8.5 pH7.0-8.5 pH6.5-8.5pH7.0-8.5 pH6.5-7.5 Heavy metal effects: activation Co, Mn Co, Mn, CaCo, Mn Co, Mn, Ca Co Heavy metal effects: strong inhibition Sn, Zn Sn,Zn Sn, Zn Sn, Zn Sn, Zn Reduction reaction product Only SIS → SI OnlySIS → SI Only SIS → SI Only SIS → SI Only SIS → SI Km values forscyllo-inosose 9.2 mM 2.6 mM 9.8 mM 3.1 mM 9.1 mM Substrate specificRelative activity SI, MI, DCI, EI SI, MI, DCI, EI, LCI SI, MI, DCI, EISI, MI, DCI, EI, SI, DCI (70% or more) LCI Substrate specific Relativeactivity LCI, MuI MuI LCI, MuI MuI MI (20 to 70%) Substrate specificRelative activity AI, NI AI, NI AI, NI AI, NI EI, LCI, MuI, (less than20%) AI, NI Abbreviations SIS: Scyllo-inosose, SI: Scyllo-inositol, MI:Myo-inositol, DCI: D-chiro-inositol, LCI: L-chiro-inositol, EI:Epi-inositol, MuI: Muco-inositol, AI: Allo-inositol, NI: Neo-inositol

TABLE 6 Homology in the amino acid sequences of various scyllo-inositoldehydrogenase E.coli.ydgJ----------MSDNIRVGLIGYGYASKTFHAPLI----AGTPGQELAVIS---SSDETKV (SEQ ID NO:2) X.camp.Xcc3438----------MPKPFNLAVYGYGYVGRTFHAPLI----ASTPGLQLHSVV---SSKPQQP (SEQ ID NO:14) B.sub.BG14057-MITLLKGRRKVDTIKVGILGYGLSGSVFHGPLL----DVLDEYQISKIM---TSRTEEV (SEQ ID NO:8) A.tume.Atu4375--MSSATKKFDSRRIRLGMVGGGQGAFIGAVHRI----AARLDDRYELVAGALSSDPARA (SEQ ID NO:4) AB10121Atu4375--MSSAFKKFDSRRIRLGMVGGGQGAFIGAVHRI----AARLDDRYELVAGALSSDPARA (SEQ ID NO:10) A.tume.Atu3234MAIEGKTTDVANKRIRLGMVGGGSGAFIGGVHRM----AARLDNRFDLVAGALSSTPEKS (SEQ ID NO:6) AB10121Atu3234MAIEGKTTDKANKRIRLGMVGGGSGAFIGGVHRM----AARLDNRFDLVAGALSSTPEKS (SEQ ID NO:12) AB10281SIDH1---------MTKRKLRIGLIGSG---FMGRTHAFGYSTASRVFDLPFQPELTCLADISDE (SEQ ID NO:28)               ::::::* *          :          .         .   .E.coli.ydgJ KADYIPTVIVYSE---------PKHLFHDPNIDLIVIPTPNDTHFPLAKAALEAGKHVVVX.camp.Xcc3438QADFREVRVLPD----------LEAALAPPALDAVVIATPNQTHAPMALQALAAGKHVLVB.sub.BG14057KRDFPDAEVVHE----------LEEITNDPAIELVIVTTPSGLHYEHTMACIQAGKHVVMA.tume.Atu4375AASATLLGIAPERSYASFEDMAATEAGREDGIEAVAIVTPNHLHFAPSKAFLEAGIHVICAB10121Atu4375AASATLIGIAPERSYASFEEMAAAEAGRDDGIEAVAIVTPNHLHFAPSKAFLEAGIHVICA.tume.Atu3234LASGRELGLDSERPYGSFEEMAEKEALREDGIEAVAIVTPNHVHYPAAKAFLERGIHVICAB10121Atu3234LASGRELGLDPERCYGSFEEMAEKEALREDGIEAVAIVTPNHVHYPAAKAFLERGIHVICAB10281SIDH1AAAKAADALGFARSTSDWRTLV-----NDPEIDVVNITAPNAFHKEMALAAIAAGKHVYC        :                   :  :: : : :*.  *   : : :. * **: E.coli.ydgJDKPFTVTLSQARELDALAKSLGRVLSVFHNRRWDSDFLTLKGLLAEGVLGEVAYFESHFDX.camp.Xcc3438DKPFALDAAQARTVVDAAAEAGKIVSVFQNRRWDADFLTVRRLIEDGQLGEVVEFHSHFDB.sub.BG14057EKPMTATAEEGETLKRAADEKGYLLSVYHNRRWDNDFLTIKKLISEGSLEDINTYQVSYNA.tume.Atu4375DKPVTATLEEAKALAGIVRASDSLFVLTHNYTGYAMLRQMREMIAEGAIGKLRHVQAEYAAB10121Atu4375DKPVTATLEEAKALAEIVRASDSLFVLTHNYTGYAMLRQMRQMVADGAIGKLRHVQAEYAA.tume.Atu3234DKPLTSNLEDAKKLKDVADKADALFILTHNYTGYPMVRHARELVEAGALGNIRLVQMEYPAB10121Atu3234DKPLTSNLEDAKKLKDVADKADALFILTHNYTGYPMVRHARELVESGALGTIRLVQMEYPAB10281SIDH1EKPLAPLAADAREMAEAAEAKGVKTQVGFNYLCNPMLALARDMIAAGELGEIRGYRGLHA :**.: ..:.. :   .   . .  :  *      .   : ::  * : .:   . E.coli.ydgJRFRP--------QVRDRWREQGGP--GSGIWYDLAPHLLDQAITLFG-LPVSM---TVDLX.camp.Xcc3438RYRP--------QVRDRWRESDIP--GAGLWYDLGPHLLDQALQLFG-MPQAI---SADLB.sub.BG14057RYRP--------EVQARWREKEGT--ATGTLYDLGSHIIDQTLHLFG-MPKAV---TANVA.tume.Atu4375QDWLTEAVEKTGAKGAEWRTDPSRSGAGGAIGDIGTHAFNAAAFVTGEIPSSL---YADLAB10121Atu4375QDWLTEAVEKTGAKGAEWRTDPSRSGAGGAIGDIGTHAFNAAAFVTGEIPKSL---YADLA.tume.Atu3234QDWLTEAVEQTGAKQAVWRTDPAQSGVGGSTGDIGTHAYNLGCFISGLEADEL---AADVAB10121Atu3234QDWLAEPIEQTGAKQAVWRTDPAQSGAGGSTGDIGTHAYNLGCFISGLEVDEL---AADVAB10281SIDH1EDYMADA-----SSPFTFRLDPA---GGGALADIGSHALATAEFLMGPAAGAITQVMGDC:                ::   .     *   *:.:*  .    : *  .  :    .:: E.coli.ydgJAQLRPGA----------QSTDYFHAILSYPQR--------RVILHGTMLAAAESARYIVHX.camp.Xcc3438QRQRTQA----------RSDDYFNVVLRYPRL--------RVILHAGSLVADGSLRFAVHB.sub.BG14057MAQRENA----------ETVDYFHLTLDYGKL--------QAILYGGSIVPANGPRYQIHA.tume.Atu4375TSFVPGR----------QLDDSANILLRYDSG-----AKGMLWASQIAVGNENALSLRVYAB10121Atu4375TSFVPGR----------QLDDSANILLRYESG-----AKGMLWASQIAVGNENALSLRVYA.tume.Atu3234HTFVEGR----------RLDDNAHVMMRFKPKGGKQPARGMLWCSQVAVGHENGLKIRLYAB10121Atu3234HTFVEGR----------RLDDNAHVMLRFKPKGGKQPAKGLLWCSQVAVGHENGLKVRVYAB10281SIDH1VTVIKTRPDGKGGTRAVEVDDIGRALLRFENG-----ATGSVEGNWIATGRTMQHDFEVY     .           .  *  :  : :                   :    .    :: E.coli.ydgJGSRGSYVKYGLDPQEERL--KNGERLP-----QEDWGYDMRD--GVLTRVEGEERVEETLX.camp.Xcc3438GTRGSYLKHGADTQEDQL--RAGRRPG-----TAGWGMDPLP--GTLTRVDDEGRVHTHQB.sub.BG14057GKDSSFIKYGIDGQEDAL--RAGRKPE-----DDSWGADVPEFYGKLTTIRGSDKKTETIA.tume.Atu4375GDKGGLEWHHRVPDELWF--TPYGEPKRLITRNGAGAGAAANRVSRVPSGHPEGYLEGFAAB10121Atu4375GEKGGLEWHHRVPDELWF--TPYGEPKRLITRNGAGAGAAANRVSRVPSGHPEGYLEGFAA.tume.Atu3234GDKAGLEWTQADPNYLWF--TKLGEPKQLITRGGAGAGAAAARVTRIPSGHPEGYLEAFAAB10121Atu3234GDKAGIEWTQADPNYLWF--TKLGELKQLITRGGAGAGAAAARVTRIPSGHPEGYLEAFAAB10281SIDH1GTKGALAFTQQRFNELHFFSSTDARGRKGFRRIEAGPEHAPYGLFCVAPGHQLGFND--- *...         ..    .           .         :.  . . E.coli.ydgJLT-VPGNYPAYYAAIRDALNGDGENPV-PASQAIQVMELIELGIESAKHRATLCLA----X.camp.Xcc3438PDGVPGDYRHCYAAFRDAMAGTAPPPV-SAADAVRLMELLELAQRGAALGQVLWLEGNSSB.sub.BG14057PS-VNGSVITYYRKIAESIREGAALPV-TAEEGINVIRIIEAAMESSKEKRTIMLEH---A.tume.Atu4375TI-YREAADAIIAKREGETAAGEVIYP-GMEDGLAGLAFIDAAVRSSQ-TSTWVGIDI--AB10121Atu4375TI-YREAADAIIAKREGKAAAGEVIYP-GMEDGIAGLAFIDAAVRSSQ-TSTWINIDI--A.tume.Atu3234TI-YTEAAHAIEARRTGSALDKAVIYP-TVDDGVKGVAFVTACIESGKKNGGWVKL----AB10121Atu3234TI-YTEAAHAIEARRTGSVLDKAVIYP-TVDDGVKGVAFVTACIESGKKNGVWVKL----AB10281SIDH1-------LKAIEVARYLEALAGHHPEPFNFRAGLRIQTLVETIHASS-KSAAWRDVPTDK          .:::  . ::   ..:.   .:     ::     :      : E.coli.ydgJ----------- X.camp.Xcc3438 D---------- B.sub.BG14057 -----------A.tume.Atu4375 ----------- AB10121Atu4375 ----------- A.tume.Atu3234----------- AB10121Atu3234 ----------- A810281SIDH1 LQAKSRQHEKA

Example 15 Production of Scyllo-inositol Using the Enzyme of the PresentInvention

The production method of the present invention requires two kinds ofenzymes, myo-inositol 2-dehydrogenase and the enzyme of the presentinvention. Herein, there will be shown an example using a recombinantenzyme of myo-inositol 2-dehydrogenase that is a product of BG10669 genederived from Bacillus subtilis 168 ATCC23857 and a recombinant enzyme ofthe enzyme of the present invention encoded by DNA of the presentinvention (ydgJ gene derived from Escherichia coli K12: SEQ ID NO: 1).

First, to obtain a recombinant enzyme of myo-inositol 2-dehydrogenasethat is a product of BG10669 gene derived from Bacillus subtilis 168ATCC23857, the following experiment was performed. PCR was performedusing primers having the following sequences for cloning BG10669 genederived from Bacillus subtilis 168 for expressing it as a recombinantenzyme.

SEQ ID NO: 25: BG10669-F 5′-ttgggatccgatgagtttacgtattggcgtaattg-3′ SEQID NO: 26: BG10669-R 5′-aaactgcagttagttttgaactgttgtaaaagattgata-3′

For PCR, Ex taq reaction solution from Takara Shuzo Co., Ltd. was used,and a solution having a composition of 5 μl of 10× Takara ExTaq Buffer,4 μl of dNTP mixture, 30 ng of a template DNA, 1 μl of each 10 μM primersolution, and 0.5 μl of Takara ExTaq was prepared by adding water sothat it has a volume of 50 p. 1, followed by layering 30 μl of mineraloil. For reaction, a cycle of three steps: denaturation (94° C., 30seconds), annealing (53° C., 1 minute), and elongation (72° C., 1minute), was repeated 35 times using PCR Amplifier (ASTEC Co., Ltd.,PC-700). The above-described PCR amplified a DNA fragment having a sizeof about 1.0 kbp. After the reaction, the layered mineral oil wasextracted with 0.3 ml of hexane, and an operation of removing the hexanelayer was repeated three times, followed by reduction of the pressurefor 1 minute, to thereby remove the mineral oil. From 50 p. 1 of thethus-obtained reaction solution, PCR fragment was purified usingGENECLEAN (Bio101). Specifically, 300 μl of the NaI solution included inthe kit was added and mixed, and 10 p. 1 of a glass beads solution wasadded and mixed. The mixture was allowed to stand at 4° C. for 15minutes and centrifuged to precipitate glass beads to which the DNAfragment was absorbed, and then the supernatant was removed. 500 μl ofNew wash solution included in the kit was further added to suspend theglass beads, and the mixture was centrifuged to remove the supernatant.The washing operation using the New wash solution was repeated threetimes. Next, the glass beads were dried under reduced pressure, and 15μl of sterilized water was added to suspend them. The mixture was heatedto 55° C. for 15 minutes and centrifuged, to thereby yield 12 μl of thesupernatant containing DNA fragment.

An operation of inserting the purified DNA fragment into an expressionvector was performed as follows. Specifically, 0.5 μg of an expressionplasmid (pUC118: manufactured by Takara Shuzo Co., Ltd.)), 1 μl of eachof restriction enzymes BamHI and PstI from Takara Shuzo Co., Ltd., and 2μl of 10×K buffer, which is a restriction enzyme buffer from TakaraShuzo Co., Ltd., were added to 10 μl of the DNA fragment solution, andsterilized water was added so that the mixture has a volume of 20followed by mixing. The reaction solution was allowed to react at 36° C.for 2 hours. After the reaction of the restriction enzymes, the DNAfragment and expression vector were isolated with GENECLEAN and ligatedwith each other. Specifically, 300 μl of the NaI solution included inthe kit was added to 20 μl of the restriction enzyme reaction solutionand mixed, and then 10 μl of the glass beads solution was added andmixed. The mixture was allowed to stand at 4° C. for 15 minutes and thencentrifuged to precipitate glass beads to which the DNA fragment andexpression vector were adsorbed, and the supernatant was removed. Then,500 μl of the New wash solution included in the kit was added to suspendthe glass beads, and the suspension was centrifuged to remove thesupernatant. The washing operation using the New wash solution wasrepeated three times. Next, the glass beads were dried under reducedpressure, and after the drying, 15 μl of sterilized water was added tosuspend them. The mixture was heated to 55° C. for 15 minutes andcentrifuged, to thereby yield 12 μl of the supernatant containing theDNA fragment and expression vector. The procedure removed small DNAfragments generated by the restriction enzymes that have sizes of about50 by or less, to thereby yield the DNA fragment of interest andexpression vector.

10 μl of a Takara Ligation kit-I solution (Takara Shuzo Co., Ltd.) wasadded to 10 μl of the thus-prepared solution, and the mixture wasallowed to react at 16° C. for 1 hour. The solution was used totransform competent cells (Takara Shuzo Co., Ltd.: DH5α). Specifically,5 μl of a ligation reaction solution was added to 60 μl of a competentcell solution unfreezed at 4° C. and mixed, and left for 30 minutes at0° C., at 42° C. for 45 seconds and at 0° C. for 2 minutes. 500 μl ofSOC solution (2% bacto-tryptone, 0.5% yeast extract, 10 mM NaCl, 20 mMglucose, 10 mM MgSO₄, and 10 mM MgCl₂) were added thereto, followed byrecovery culture at 36° C. for 1 hour. 100 μl of the culture solutionwas applied on an LB agar medium (1% bacto-tryptone, 0.5% yeast extract,1% NaCl, pH 7.0, 1.5% agar) containing 50 μg/ml ampicillin, 40 μg/mlX-gal (5-Bromo-4-Chloro-3-Indolyl-β-D-Galactoside), and 1 mM IPTG(thiogalactopyranoside). Culture was further performed at 37° C. for 16hours. The culture yielded Escherichia coli transformed by introducingthe above-described plasmids as white colonies, and the colonies wereselected. The thus-separated colonies of transformed Escherichia coliwere cultured in an LB liquid medium containing ampicillin (50 μg/ml).From the cultured cells of transformed Escherichia coli, the plasmid DNAwas separated and purified using a plasmid purification kit (QIA filterPlasmid Midi Kit, QIAGEN). The thus-obtained plasmid DNA was confirmedto have a DNA fragment having a size of about 1.0 k bp, whichcorresponds to the BG10669 gene of interest.

Next, to confirm the scyllo-inositol 2-dehydrogenase activity, the cellsisolated as colonies were transferred into 30 bottles of 100 ml of an LBmedium (1% bacto-tryptone, 0.5% yeast extract, 1% NaCl, pH 7.0)containing 50 μg/ml ampicillin, and they were cultured at 36° C. for 7hours. 0.3 ml of 200 mM thiogalactopyranoside solution was added to each100 ml of the culture solution, and the cells were further cultured at36° C. for 3 hours. After completion of the culture, the cells werecollected by centrifugation and washed with physiological saline once.Then, the washed cells were suspended in 3 ml of 0.6% Triton X-100solution, and the cells were disrupted by ultrasonic wave at 4° C. Thesolution was centrifuged, and 84 ml of the supernatant (enzyme solution)was taken out. 36 g of ammonium sulfate was added to the supernatant tosalt out proteins at 4° C. The resultant proteins were collected bycentrifugation, and the supernatant was removed. The precipitates weredissolved in 75 ml of 20 mM Tris buffer (pH 7.0), and the solution wascentrifuged again. The supernatant was applied onto Sephadex G-25 column(Pharmacia K.K.)(400 ml) equilibrated with 20 mM Tris buffer (pH 7.0),and elution was performed with 20 mM Tris buffer (pH 7.0). Elution wasperformed with 20 mM Tris buffer (pH 7.0), and the eluate was desalted.The procedures yielded 105 ml of a crude enzyme solution of BG10669 geneproduct.

The myo-inositol 2-dehydrogenase reducing activity was measured asdescribed below. 5 μl of a reaction solution (200 mM Tris buffer (pH8.0), 2% of NADH, 1% of scyllo-inosose) was mixed with 5 μl of an enzymesolution, and the mixture was allowed to react at 36° C. for 30 min.Immediately after the reaction, 500 μl of water was added, and theabsorbance at 340 nm was measured. From the blank value of a test tubeprepared by using water instead of the enzyme solution, the decrease inthe absorbance at 340 nm was measured. The enzyme solution was dilutedas required, and the enzyme was confirmed to have an activity to reducescyllo-inosose. On the other hand, to measure an oxidation activity, 50μl of a reaction solution (1% myo-inositol or scyllo-inositol, 200 mMTris buffer (pH 8.0), 0.002% NAD⁺, 0.002% diaphorase, and 0.01%nitrotetrazolium blue) was mixed with 50 μl of an enzyme solution, andthe increase in the absorbance at 545 nm was measured at 25° C. everythree minutes using a microplate reader. The reaction rate wascalculated from the absorbance increments at the respective times. As aresult, the prepared enzyme was confirmed to have an activity to oxidizemyo-inositol but have no activity to oxidize scyllo-inositol.

The thus-prepared myo-inositol 2-dehydrogenase enzyme solution and thescyllo-inositol dehydrogenase crude enzyme solution prepared in Example11 (105 ml of the enzyme solution prepared from 3 L of a culturesolution (30-fold scale)) were used to perform a reaction to convertmyo-inositol into scyllo-inositol. To prepare a reaction solution, 200 gof myo-inositol, 70 ml of 5% of scyllo-inosose, 130 mg of CoCl₂, and 250mg of MgSO₄.7H₂O were mixed, and the volume was adjusted to 750 ml byadding water, followed by heating up to 50° C. to dissolve myo-inositol.The solution was cooled to 36° C. and adjusted to pH 8.0 with 1N NaOHaqueous solution, and the volume was adjusted to 790 ml by adding water.105 ml of a crude enzyme solution of myo-inositol 2-dehydrogenase, 105ml of a crude enzyme solution of scyllo-inositol dehydrogenase, and 70mg of NADP⁺ were added thereto at 36° C., and the temperature of thesolution having a volume of about 1 L was kept at 36° C., followed byreaction with slow stirring. The reaction solution gradually becameacidic, so that it was adjusted to pH 8.0 with 1N NaOH. 42 hours later,crystals of scyllo-inositol were generated in the reaction solution, sothat a white reaction solution was obtained. The solution was filteredusing a filter paper to collect crystalline scyllo-inositol (wet weight73 g). 3 L of water was added to the solid, and the solid was dissolvedat 50° C. The volume of the mixture was adjusted to 4.5 L by addingwater, and the mixture was cooled to room temperature. The resultantsolution was centrifuged (8,000 rpm, 20 minutes) to remove insolublematters, and the supernatant was passed through a column filled with 100ml of strong base cation exchange resin, a column filled with 100 ml ofstrong acid anion exchange resin, and a column filled with 50 ml ofactivated carbon, in this order, to thereby yield each of an eluate.Thereafter, 500 ml of water was passed through each of the columns towash them, to thereby yield each of a washing solution. The eluate andthe washing solution were blended and concentrated.

As a result of the concentration, microcrystals began to be crystallizedas the volume of the solution became small, and the solution wasconcentrated until the weight of the contents became 130 g. Theconcentrated solution was cooled to 4° C. and allowed to standovernight. Thereafter, slurry substances were filtered, and the crystalsof scyllo-inositol on the filter paper were washed with a small amountof water, followed by drying at 105° C. for 3 hr. The resultantscyllo-inositol was white crystals (61 g), and NMR analysis and HPLCanalysis revealed that the crystals contain no other impurities and havea purity of 99% or more. The yield from myo-inositol was 31%. Meanwhile,the reaction solution that had been separated by filtration could alsobe utilized, and when 64 g of myo-inositol was dissolved therein,crystalline scyllo-inositol was further crystallized.

Example 16 Formation of Scyllo-inositol/boric Acid Complex and Study onthe Formation Condition

100 g of scyllo-inosose powder was dissolved in 500 ml of hot water, andthe solution was cooled to room temperature. Thereafter, water was addedin such a manner that the solution has a volume of 900 ml. The solutionwas adjusted to pH 7.5 with 5N NaOH aqueous solution, and water wasfurther added so as to have a volume of 1 L.

5.9 g of NaBH₄ powder was gradually added to the solution over 15minutes with stirring to perform a reduction reaction. The temperatureof the reaction solution increased up to 38° C. due to heat of thereaction. 30 minutes later, 67.5 g of boric acid and 72.2 g of NaCl weredissolved in the reaction solution which had been cooled to 32° C., tothereby prepare a solution in which the complex was formed. The pH ofthe solution was 5.9.

Next, 100 ml of the solution, in which the complex was formed and wasadjusted to pH 6.0 with 8N NaOH aqueous solution, was dispensed in a200-ml plastic container with a cover, and 100 ml of the solution, inwhich the complex was formed and was adjusted to pH 7.0 with 8N NaOHaqueous solution, was dispensed in the same way as above. Furthermore,each of 100 ml of the solutions, in which the complex was formed andeach of which was adjusted to pH 8.0, 9.0, 9.5, 10.0, 10.5, 11.0, 12.0,or 12.8, were dispensed.

In those pH-adjusted solutions, precipitates gradually began to beformed. The precipitates were separated by filtration every other day,and the filtrate was adjusted to a predetermined pH with 8N NaOH aqueoussolution and then returned to the original container. The resultantprecipitates were dried and then weighed. If all of the scyllo-inositolgenerated by reduction form a scyllo-inositol/boric acid complex and areobtained as precipitates, the weight of the precipitates would be 61.8g. Therefore, the weights of the precipitates obtained from therespective pH-adjusted solutions were integrated every other day, andvalues obtained by dividing the integrated values by theoretical yield(61.8 g) were defined as the recovery rates of the scyllo-inositol/boricacid complex precipitates.

The thus-obtained values are shown below.

The gray parts in the table show a test group of the recovery rate ofmore than 90%.

TABLE 7

As shown in Table 7, the test group of treatment at pH of 9.5 was mostsuitable for formation of precipitation of the scyllo-inositol/boricacid complex. The recovery rates of the test groups of pH 9.0, pH 9.5,and pH 10.0 reached a recovery rate of 90% or more by day 4, and it wasfound that the recovery rate became constant of 94% even if the testperiod is extended.

NMR analysis for the filtrate of the test group of pH 9.5 at day 7revealed that 5.9% (w/v) myo-inositol and about 0.2% (w/v)scyllo-inositol remained in the solution. That is, it was found that thecomplex may be taken out as precipitates by the method of the presentinvention in the case where the concentration of thescyllo-inositol/boric acid complex is 0.2% (w/v) or more.

Example 17 Method of Forming Scyllo-inositol/boric Acid Complex fromScyllo-inosose Reduction Mixture and Dissolving the Complex, and thenReleasing and Desalting Scyllo-inositol Using Ion Exchange Resin

10 g (56 mmol) of scyllo-inosose powder was dissolved in 50 ml of hotwater, and the solution was cooled to room temperature. Thereafter,water was added so as to have a volume of 90 ml. The solution wasadjusted to pH 7.5 with 5N NaOH aqueous solution, and water was furtheradded so as to have a volume of 100 ml.

0.59 g of NaBH₄ powder was gradually added to the solution over 15minutes with stirring to perform a reduction reaction. The temperatureof the reaction solution increased up to 36° C. due to heat of thereaction. 30 minutes later, 6.75 g of boric acid and 7.22 g of NaCl weredissolved in the reaction solution cooled to 31° C., to thereby preparea solution in which complex was formed. Next, the solution in whichcomplex was formed was adjusted to pH 9.5 with 5N NaOH aqueous solution,and the solution was maintained to pH 9.5 with 5N NaOH aqueous solutionby pH stat apparatus with stirring. 3 days later, precipitates containedin the solution in which complex was formed were filtered and washedwith a small amount of water, followed by drying, to thereby yield 5.71g (20.5 mmol) of a scyllo-inositol/boric acid complex.

230 ml of 1.05N hydrochloric acidic solution was added to 5.71 g of theresultant scyllo-inositol/boric acid complex, to dissolve thescyllo-inositol/boric acid complex, and thereby a dissolved solution wasobtained. The dissolved solution was 0.2N acidic solution. Next, thedissolved solution was passed through a column filled with 200 ml ofstrong acidic ion exchange resin (Duolite C20, H⁺ type, SumitomoChemical Co., Ltd.) at a flow rate of 2 ml/min, and the resultant eluatewas then passed through a column filled with 400 ml of strong base ionexchange resin (Duolite A116, OH⁻ type, Sumitomo Chemical Co., Ltd.).The resultant eluate was concentrated, to thereby yield 3.52 g (19.5mmol) of white powder. NMR analysis revealed that the white powder wasscyllo-inositol. Meanwhile, the yield of scyllo-inositol fromscyllo-inosose was 35%.

Example 18 Method of Forming Scyllo-inositol/boric Acid Complex from aScyllo-inosose Reduction Mixture and Dissolving the complex, and thenReleasing and Crystallizing Scyllo-inositol by Organic SolventPrecipitation

As a raw material, 5.71 g (20.5 mmol) of a scyllo-inositol/boric acidcomplex that had been prepared from 10 g (56 mmol) of scyllo-inososepowder in the same way as Example 17 was used.

5.71 g of a scyllo-inositol/boric acid complex was added to a 100-mlconical flask with a cover together with a stirrer, and 22.8 ml of 1.83Nhydrochloric acidic solution was added to prepare a suspension. Afterthe completion of stirring for 1 hour, 23 ml of methanol was addedthereto, and the mixture was further stirred. 5 hours later, thesuspension was filtered, and the solids were washed with a small amountof methanol and dried, to thereby yield 3.58 g (20.0 mmol) of crudescyllo-inositol.

Then, 3.58 g of the resultant crude scyllo-inositol was dissolved in 230ml of water, and 20 ml of a strong acidic ion exchange resin (DuoliteC20/H⁺ type) and 40 ml of a strong base ion exchange resin (DuoliteA116/OH⁻ type) were added thereto, followed by stirring. After thecompletion of stirring for 30 minutes, the ion exchange resins wereseparated by filtration, and the resultant filtrate was concentrated, tothereby yield 3.41 g (18.9 mmol) of white powder. NMR analysis revealedthat the white powder was scyllo-inositol. Meanwhile, the yield ofscyllo-inositol from scyllo-inosose was 34%.

Example 19 Method of Reducing Scyllo-inosose, and then DirectlyReleasing and Crystallizing Scyllo-inositol

5 g (28 mmol) of scyllo-inosose powder was dissolved in 40 ml of hotwater, and the solution was cooled to room temperature. The solution wasadjusted to pH 7.5 with 5N NaOH aqueous solution, and water was furtheradded so as to have a volume of 45 ml.

0.29 g of NaBH₄ powder was gradually added to the solution over 15minutes with stirring to perform a reduction reaction. The temperatureof the reaction solution increased up to 37° C. due to heat of thereaction. 30 minutes later, the reaction solution which had been cooledto 30° C. was adjusted to pH 1.0 with 5N hydrochloric acid. Thereafter,water was added so as to have a volume of 50 ml, to thereby prepare 0.1Nacidic solution. Next, 25 ml of methanol was added to the solution withstirring. 10 minutes later, the solution gradually began to becomeopaque, and the suspension was further stirred for 24 hours. 24 hourslater, the suspension was filtered, and washing was performed with asmall amount of methanol, followed by drying, to thereby yield 1.55 g(8.6 mmol) of crude scyllo-inositol.

Then, 1.55 g of the resultant crude scyllo-inositol was dissolved in 120ml of water, and 10 ml of a strong acidic ion exchange resin (DuoliteC20/H⁺ type) and 20 ml of a strong base ion exchange resin (DuoliteA116/OH⁻ type) were added, followed by stirring. After the completion ofstirring for 30 minutes, the ion exchange resins were separated byfiltration, and the resultant filtrate was concentrated, to therebyyield 1.51 g (8.3 mmol) of white powder. NMR analysis revealed that thewhite powder was scyllo-inositol. Meanwhile, the yield ofscyllo-inositol from scyllo-inosose was 30%.

INDUSTRIAL APPLICABILITY

According to the present invention, scyllo-inositol that is available asa drug can be directly produced from inexpensive myo-inositol only bymicroorganism conversion or enzymatic reaction, and scyllo-inositol canbe produced efficiently. Meanwhile, the production method of the presentinvention has an advantage of hardly generating isomers.

When NAD⁺-independent myo-inositol 2-dehydrogenase of the presentinvention is used, scyllo-inosose may be produced without adding NAD⁺ toa reaction solution. Moreover, high purity of scyllo-inositol may beobtained easily and efficiently by reduction of the resultantscyllo-inosose.

According to the present invention, a scyllo-inositol/boric acid complexcan be efficiently formed from a mixture that contains scyllo-inositoland neutral sugars other than scyllo-inositol, and high purity ofscyllo-inositol can be efficiently obtained from the resultantscyllo-inositol/boric acid complex by easy operation.

1. A method of producing scyllo-inositol comprising: allowing amicroorganism capable of converting myo-inositol into scyllo-inositoland belonging to the genus Acetobacter or Burkholderia to react withmyo-inositol in a solution containing myo-inositol to produce andaccumulate scyllo-inositol in the solution; and collecting thescyllo-inositol from the solution.
 2. The method according to claim 1,wherein the solution containing myo-inositol is a liquid mediumcontaining myo-inositol, and the microorganism is allowed to react withmyo-inositol by culturing the microorganism in the liquid medium.
 3. Themethod according to claim 1, wherein cells obtained by culturing themicroorganism are allowed to react with myo-inositol in the solution. 4.The method according to claim 1, wherein the microorganism is amicroorganism belonging to Acetobacter cerevisiae, Acetobacter malorum,or Burkholderia andropogonis.
 5. The method according to claim 1,wherein the microorganism is Acetobacter sp. AB10281 strain (FERMBP-10119) or a mutant strain thereof.